Performance and Verification of a Real-Time PCR Assay Targeting the gyrA Gene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae

Abstract

cIn the United States, 19.2% of Neisseria gonorrhoeae isolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive for N. gonorrhoeae by the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions. The sexually transmitted disease gonorrhea is the second most commonly reported notifiable disease in the United States, with 350,062 cases reported in 2014 (1). The etiologic agent of the disease, Neisseria gonorrhoeae, has become a major concern worldwide due to the high prevalence of resistance to antimicrobials that were at one time part of the standard treatment for the disease, including the sulfonamides, penicillins, narrow-spectrum cephalosporins, tetracyclines, macrolides, and fluoroquinolones (2). In the United States, multidrug-resistant N. gonorrhoeae is categorized as an urgent threat to public health by the Centers for Disease Control and Prevention (CDC) (3). As a result of emerging resistance, the CDC has issued several changes to the treatment guidelines for gonococcal infections, which included the removal of single-dose oral cephalosporins from the 2012 recommendations due to a significant decline in cefixime susceptibility among the gonococci (4). The current CDC treatment guideline now recommends the use of two drugs: a single intramuscular injection of ceftriaxone followed by a single dose of azithromycin administered orally (5).In the United States, the prevalence of fluoroquinolone-resistant gonococci has declined from its peak in 2007, when the CDC ceased recommending ciprofloxacin as empirical treatment for gonococcal infections (6). For example, the prevalence of ciprofloxacin-resistant N. gonorrhoeae isolates in San Francisco decreased dramatically from 44% in 2006 to 9.6% in 2009 (7), which coincided with these changes to national treatment guidelines. Currently, the proportion of U.S. isolates resistant to ciprofloxacin (MIC, Ն1.0 g/ml) is at 19.2% (CDC, unpublished data), suggesting that ciprofloxacin may still be a viable option in some cases for treatment of infections, the majority of which are caused by ciprofloxacin-susceptible isolates. However, as few laboratories routinely perform culture and susceptibility testing for N. gonorrhoeae, a molecular assay is needed to differentiate patients infected with a susceptible strain from those infected with a resistant strain. Resistance to ciprofloxacin is conferred by mutation in the Ser91 codon of the gyrA gene in Ͼ99% of all resistant isolates investigated to date, making this mutation an attractive target for prediction of ciprofloxacin susceptibility (8). In this study, we evaluated a real-time (RT)-PCR assay of the gyrA gene that was coupled with high-resol...

Genomic Epidemiology of Gonococcal Resistance to Extended-Spectrum Cephalosporins, Macrolides, and Fluoroquinolones in the United States, 2000–2013

“…Here, we extend our analysis to include 1102 gonococcal isolates drawn from across the United States and over 14 years (2000–2013) and resistance to 3 of the most clinically relevant antimicrobial classes: the ESCs, including cefixime and ceftriaxone; macrolides, specifically azithromycin; and fluoroquinolones, specifically ciprofloxacin. We focus on these 3 classes because the current recommendation for treatment of gonorrhea is dual therapy with ceftriaxone and azithromycin [2] and because advances in development of molecular diagnostic tests may prompt reconsideration of treatment with fluoroquinolones [3, 4], which has not been recommended since the population prevalence of quinolone resistance exceeded 5% [5]. …”

Why Can't We Just Use PCR? The Role of Genotypic versus Phenotypic Testing for Antimicrobial Resistance Testing

“…Although not widely available for clinical use, genotypic screening for the gyrA gene in Neisseria gonorrhoeae has proven to be highly predictive of ciprofloxacin resistance [22]. Additional screening for extended-spectrum cephalosporins by targeting penA mosaic XXXIV yielded high sensitivity and specificity (97% and 100%, respectively) [23].…”

Real-Time PCR Targeting the penA Mosaic XXXIV Type for Prediction of Extended-Spectrum-Cephalosporin Susceptibility in Clinical Neisseria gonorrhoeae Isolates

“…As such, there is a need for molecular AST assays that can predict N. gonorrhoeae susceptibility to antimicrobials commonly used to treat gonococcal infections directly from clinical specimens. We recently developed a real-time PCR with fluorescence resonance energy transfer (FRET) hybridization probes to predict susceptibility for ciprofloxacin by targeting the gyrA Ser91 mutation, with 100% correlation between genotype and in vitro susceptibility to ciprofloxacin for N. gonorrhoeae (16,17). In this study, we sought to develop an assay to predict decreased susceptibility to ESCs in N. gonorrhoeae, to be performed as a multiplex test with gyrA to allow for simultaneous prediction of susceptibility to both ESCs and ciprofloxacin.…”

“…CPD MICs were also available for the CDC isolates. The isolates from the Neisseria Reference Laboratory were recovered between 2011 and 2014 from cultures obtained from urine and pharyngeal, rectal, urethral, and cervical specimens of gonococcal infections, and they were previously used to evaluate the singleplex gyrA assay (16). Thirty-two isolates were ciprofloxacin susceptible (MIC, Յ 0.012 g/ml), and 118 isolates were ciprofloxacin resistant (MIC range, 1 to Ͼ16 g/ml) based on CLSI breakpoints (28).…”