title: Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae.
abstract: The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.
authors: Bacteriology and Enteric Diseases Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors: S W
authors: SW
authors: Peterson
authors: Bacteriology and Enteric Diseases Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors: I
authors: I
authors: Martin
authors: Bacteriology and Enteric Diseases Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors: W
authors: W
authors: Demczuk
authors: Bacteriology and Enteric Diseases Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors: A
authors: A
authors: Bharat
authors: British Columbia Centres for Disease Control Public Health Microbiology and Reference Laboratory, Vancouver, British Columbia, Canada.
authors: L
authors: L
authors: Hoang
authors: Cadham Provincial Laboratory, Winnipeg, Manitoba, Canada.
authors: J
authors: J
authors: Wylie
authors: Public Health Ontario Laboratories, Toronto, Ontario, Canada.
authors: V
authors: V
authors: Allen
authors: Laboratoire de Santé Publique du Québec, Ste-Anne-De-Bellevue, Québec, Canada.
authors: B
authors: B
authors: Lefebvre
authors: Provincial Laboratory for Public Health, Edmonton, Alberta, Canada.
authors: G
authors: G
authors: Tyrrell
authors: Saskatchewan Disease Control Laboratory, Regina, Saskatchewan, Canada.
authors: G
authors: G
authors: Horsman
authors: Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia, Canada.
authors: D
authors: D
authors: Haldane
authors: Dr. G.L. Dumont Hospital, Moncton, New Brunswick, Canada.
authors: R
authors: R
authors: Garceau
authors: Centre for Communicable Diseases and Infection Control, Public Health Agency of Canada, Ottawa, Ontario, Canada.
authors: T
authors: T
authors: Wong
authors: Bacteriology and Enteric Diseases Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada michael.mulvey@phac-aspc.gc.ca.
authors: M R
authors: MR
authors: Mulvey
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002170:Canada; D002511:Cephalosporins / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D005260:Female; D005798:Genes, Bacterial; D005819:Genetic Markers; D060005:Genotyping Techniques / Q000379:methods*; D006069:Gonorrhea / Q000382:microbiology; D006801:Humans; D008297:Male; D008828:Microbiological Techniques / Q000379:methods; D009344:Neisseria gonorrhoeae / Q000187:drug effects* / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D005819:Genetic Markers