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.

Figures

FIG. 1.

Ciprofloxacin MICs and melting-curve temperature for 96 N. gonorrhoeae-positive urine isolates. Of 96 specimens, 53 had a melting-curve temperature of 66°C, 42 had a melting-curve temperature of 56°C, and 1 did not amplify. DN, did not amplify; GyrA Mut, specimens with a melting-curve temperature of 56°C ± 1°C corresponding to a mutant genotype; GyrA WT, specimens with a melting-curve temperature of 66°C ± 1°C corresponding to a wild-type genotype. MICs are presented (in mg of ciprofloxacin/dl) as determined by the regional GISP laboratory in Seattle, WA.

FIG. 2.

Melting-curve analysis of 14 urine specimens with known MICs for ciprofloxacin, including six resistant isolates, seven susceptible isolates, and one negative control. Resistant isolates were defined as those with an MIC of ≥0.125 μg/ml by culture and susceptibility testing. Sensitive isolates were defined as those with an MIC of