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

Abstract

Neisseria gonorrhoeae isolates with decreased susceptibility to extendedspectrum cephalosporins (ESCs) are increasing. We developed an assay to predict N. gonorrhoeae susceptibility to ESCs by targeting penA mosaic XXXIV, an allele prevalent among U.S. isolates with elevated ESC MICs. The assay was 97% sensitive and 100% specific for predicting at least one ESC MIC above the CDC alert value among clinical isolates, and it could be multiplexed with a previously validated gyrA PCR to predict ciprofloxacin susceptibility.

Genotypic and Phenotypic Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae: a Cross-Sectional Study of Isolates Recovered from Routine Urine Cultures in a High-Incidence Setting

“…Our study adds to a growing body of literature demonstrating strong associations between specific genotypic markers and resistance to several antibiotics [i.e. , bla TEM-1b for penicillin, tet (M) for tetracyclines, and gyrA with or without parC for quinolones], suggesting that additional genetic targets—via a targeted or whole-genome sequencing approach—may enable the effective use of antimicrobials that are no longer appropriate for use as empirical therapy (27–29). However, our identification of one ciprofloxacin-resistant isolate lacking any known quinolone resistance determinants highlights the current limitations of this approach and the need for ongoing efforts to interrogate the resistome of N.…”

Using the genetic characteristics of Neisseria gonorrhoeae strains with decreased susceptibility to cefixime to develop a molecular assay to predict cefixime susceptibility

“…Previously, we developed a polymerase chain reaction-based assay using high resolution melt analysis to predict N. gonorrhoeae susceptibility to ceftriaxone and cefixime by targeting the penA 34 type. 68 While that assay showed greater than 98% sensitivity in predicting cefixime-decreased susceptibility in North America, where the penA 34 is the most common penA type, 27 , 50 the assay is limited by its inability to identify cefixime-decreased susceptibility as a result of N. gonorrhoeae strains with non-mosaic penA or other non-34 mosaic penA patterns. As N. gonorrhoeae strains with various non-34 penA types account for many of the cefixime-resistant strains reported in Asia and Europe, it is likely only a matter of time for such strains to emerge in other parts of the world, including in the USA.…”

“…A molecular assay that detects any penA alterations at amino acid positions 375-377, 501, 542 or 551 would be the most parsimonious assay to predict decreased susceptibility to cefixime. That modified assay would be more effective in predicting cefixime susceptibility than the assay we previously developed, 68 with greater sensitivity for diverse international isolates. Based on the molecular characteristics of all reported strains with decreased susceptibility to cefixime as recorded in Table 1, the estimated sensitivity for the proposed assay would be 99.5% (413 out of 415).…”

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]. This is particularly relevant, as a majority of gonococcal infections are diagnosed by molecular methods, and recovery of organisms for AST is uncommon.…”