Improved determination of Neisseria gonorrhoeae gyrase A genotype results in clinical specimens

Abstract

Background The emergence of drug-resistant Neisseria gonorrhoeae has prompted the development of rapid molecular assays designed to determine antimicrobial susceptibility. One common assay uses high-resolution melt analysis to target codon 91 of the gyrase A gene (gyrA) to predict N. gonorrhoeae susceptibility to ciprofloxacin. Methods We extracted DNA from remnant clinical specimens that had previously tested positive for N. gonorrhoeae using the Aptima Combo 2 for CT/NG assay (Hologic, San Diego, CA, USA). We selected DNA extracts from specimens with indeterminate, WT and mutant gyrA genotype results from a previous study using high-resolution melt analysis to detect the gyrA codon 91 mutation. We re-tested those specimens using the recently CE-marked ResistancePlus GC (beta) assay (SpeeDx, Sydney, Australia). Results Of 86 specimens with indeterminate gyrA genotypes on high-resolution melt analysis, the ResistancePlus GC (beta) assay (SpeeDx) identified 30 (35%) WT, 22 (26%) mutant and 34 (40%) indeterminate gyrA genotypes. Conclusions The ResistancePlus GC (beta) assay showed improved N. gonorrhoeae gyrA genotype determination compared with a prior gyrA genotypic high-resolution melt assay.

Genomic Analysis of the Predominant Strains and Antimicrobial Resistance Determinants Within 1479 Neisseria gonorrhoeae Isolates From the US Gonococcal Isolate Surveillance Project in 2018

“…Sixty percent of GC specimens show CIP susceptibility, although these numbers fluctuate per year. With the previously known significant correlation between CIP R and the GyrA S91F variant (also observed in our data-Supplemental Table 1B, http://links.lww.com/OLQ/A682), diagnostics are being developed for rapid detection of CIP susceptibility 26 to present CIP as an alternative personalized treatment. 27 With high-resolution data from WGS and increased understanding of GC AMR determinants, the goal is to develop similar diagnostic tests which may be used for other antibiotics.…”

Reflex Detection of Ciprofloxacin Resistance in Neisseria gonorrhoeae by Use of the SpeeDx ResistancePlus GC Assay

“…These samples were stored as per the manufactures instructions at room temperature prior to testing on the Aptima assay, and were all tested within 14 days post-NAAT. PCR testing for N. gonorrhoeae and the GyrA S91F mutation was performed on 5 μl of extracted DNA using the previously described ResistancePlus GC assay (SpeeDx Pty Ltd., Sydney, Australia) ( 8 , 9 ) on a LightCycler 480 II (LC480 II; Roche, Switzerland). Briefly, the assay reports detection across five channels using the following targets: (i) detection of the N. gonorrhoeae opa gene; (ii) detection of the N. gonorrhoeae porA gene; (iii) detection of gyrA S91 (wild type); (iv) detection of gyrA S91F; and (v) an internal control to monitor extraction efficiency and qPCR inhibition.…”

Bridging the gap between development of point-of-care nucleic acid testing and patient care for sexually transmitted infections

“…Novel PCR probe design. The aforementioned ResistancePlus® assay uses a novel PCR probe technology called PlexZyme, which is based on MNAzyme qPCR 197 and is currently commercialized by SpeeDx (Australia), and has been used to detect NG and its ciprofloxacin resistance gene (gyrA) 198,199 and M. genitalium and its azithromycin resistance gene (23S rRNA). 68,200 This technology uses a pair of partial nucleic acid enzymes to create a new class of qPCR probes with enhanced specificity and improved capacity for multiplexing when compared to qPCR using Taqman probes or molecular beacons, which have been empirically demonstrated.…”