Multiplex Real-Time PCR Assay for Simultaneous Identification of Neisseria gonorrhoeae and Its Ciprofloxacin Susceptibility Status

Abstract

A real-time PCR (RT-PCR) assay was designed for the simultaneous identification of and its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) in of The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254 isolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non- species isolates. The performance of the primers was validated using genomic DNA from 100 different isolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non- isolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of the isolates and 23 non- isolates. These primers detected and its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification of and its ciprofloxacin susceptibility status.

Trends and Risk Factors for Antimicrobial-Resistant Neisseria gonorrhoeae , Melbourne, Australia, 2007 to 2018

“…Recently, several PCR-based molecular assays for the direct detection of ciprofloxacin resistance in N. gonorrhoeae have been developed, including a commercially available assay (20, 21). These assays offer considerable promise in enhancing AMR surveillance in the absence of bacterial culture, i.e., when clinical specimens only undergo NAAT and could (depending on whether clinically relevant turnaround times can be achieved) be used to guide individualized treatment regimens based on their resistance profile.…”

Molecular Characterization of Markers Associated With Antimicrobial Resistance in Neisseria gonorrhoeae Identified From Residual Clinical Samples

“…Faster molecular approaches at the point-of-care (POC) are needed which can simultaneously identify NG infections and predict susceptibility to guide antimicrobial stewardship. 16 The ideal method for AMR testing in NG would be to develop a rapid, phenotypic assay that would directly determine the AMR profile of the NG organism present in a given sample. However, because that technology does not yet exist, and given the decreasing number of clinical isolates available for culture due to the increase in NAAT testing for NG infections, molecular methods represent the best intermediary step until a rapid, phenotypic assay is developed.…”

“…15 Furthermore, an assay has recently been developed, which can simultaneously detect NG and its ciprofloxacin susceptibility status as a tool to increase surveillance for ciprofloxacin resistance. 16 Implementation of molecular assays for detection of AMR NG could better define the epidemiology of AMR NG, guide antimicrobial treatment options, and provide alternatives to traditional susceptibility methods. In the present study, real-time PCR assays were employed to detect NG and AMR determinants directly from residual urethral swabs.…”

Developing target product profiles for Neisseria gonorrhoeae diagnostics in the context of antimicrobial resistance: An expert consensus

“…A test that could predict AMR/susceptibility to currently available antibiotics (particularly ceftriaxone and cefixime) and new antibiotics under development for NG would be ideal, however the technical challenges associated with developing such an assay mean that other key antibiotics might need to be chosen for inclusion in the assay. Several molecular tests under development include the detection of ciprofloxacin resistance/susceptibility [59,60]; however, its utility in most African and South East Asian regions would be limited due to the high levels of resistance to this antibiotic. Whilst there is potential to develop a penicillin high level resistance test based on beta-lactamase production, the experts were concerned about how to deal with lower level or intermediate non-beta-lactamase (chromosomal) resistance to penicillin, as the predictive value of relevant markers is not fully understood, and treating people with intermediate resistance will likely drive more resistance.…”