title: Real-time PCR assay for detection of quinolone-resistant Neisseria gonorrhoeae in urine samples.
abstract: A need exists for the development of applicable surveillance tools to detect fluoroquinolone-resistant Neisseria gonorrhoeae (QRNG) in urine samples. We describe here a real-time PCR assay for detecting mutations in the Ser91 codon of the gyrA gene of N. gonorrhoeae in urine specimens. We tested 96 urine samples collected along with Gonorrhea Isolate Surveillance Project (GISP) urethral swab samples and compared the results with matched MICs of ciprofloxacin, as reported by the regional GISP laboratory. We then tested 100 urine specimens, known to be gonorrhea positive by nucleic acid amplification testing, provided by females to challenge the real-time PCR assay with urine specimens containing potentially less target DNA content than specimens from symptomatic males. With an MIC threshold of 0.125 mug of ciprofloxacin/ml, our assay correctly identified resistance in 41 of 44 (93.2%; 95% confidence interval [CI] = 81.3 to 98.6%) corresponding resistant culture specimens and correctly identified 51 of 51 (100%; 95% CI = 93.0 to 100%) susceptible specimens. One specimen did not amplify. The assay successfully amplified the gyrA amplicon and determined a susceptibility genotype in 72 of 100 (72%) urine specimens collected from female patients. We developed an assay for detecting QRNG in urine specimens that correlated well with MIC results of cultured specimens and had moderate sensitivity with urine specimens. This methodology might fulfill the need for a QRNG detection system for urine specimens, a useful characteristic in the age of nucleic acid amplification testing for gonococcal infection.
authors.affiliation: Johns Hopkins School of Public Health, Baltimore, Maryland, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Siedner
authors.forename: Mark
authors.initials: M
authors.lastname: Pandori
authors.forename: Lina
authors.initials: L
authors.lastname: Castro
authors.forename: Pennan
authors.initials: P
authors.lastname: Barry
authors.forename: William L H
authors.initials: WL
authors.lastname: Whittington
authors.forename: Sally
authors.initials: S
authors.lastname: Liska
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Klausner
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D027081:DNA Gyrase / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005260:Female; D024841:Fluoroquinolones / Q000494:pharmacology*; D006069:Gonorrhea / Q000382:microbiology*; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests; D020125:Mutation, Missense; D009344:Neisseria gonorrhoeae / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014521:Urethra / Q000382:microbiology; D014556:Urine / Q000382:microbiology*
chemical_list: D000900:Anti-Bacterial Agents; D024841:Fluoroquinolones; D027081:DNA Gyrase