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Published in final edited form as: J Microbiol Methods. 2019 Nov 21;168:105783. doi: 10.1016/j.mimet.2019.105783

Genotyping Neisseria gonorrhoeae gyrA and penA antimicrobial genes from remnant Neisseria gonorrhoeae positive Cepheid Xpert® clinical specimens – A feasibility study

S Qadir 1, O Ellis 2, EM Keizur 1, C Stafylis 1, R Cortado 3, JD Klausner 1; the Adolescent Trials Network (ATN) CARES Team
PMCID: PMC11964821  NIHMSID: NIHMS1545434  PMID: 31760050

Abstract

Neisseria gonorrhoeae (NG) has developed resistance to most antibiotics, making it increasingly difficult to treat. Previous studies have predicted antimicrobial NG susceptibility based on the antimicrobial gene target DNA gyrase subunit A (gyrA) codon serine 91 and the penicillin-binding protein 2 (penA) using Roche Cobas® and Hologic APTIMA™ clinical specimens. We studied whether similar methods could be used on remnant NG-positive Cepheid Xpert® specimens.

Keywords: Genotyping, penA, gyrA, Neisseria gonorrhoeae, Antibiotic Resistance

Neisseria gonorrhoeae (NG) is an increasingly common sexually transmitted infection and becoming more difficult to treat (Centers for Disease Control and Prevention (CDC), 2014). Globally, antimicrobial resistant NG is a major problem. The CDC has designated antimicrobial resistant NG as one of the three most urgent threats to public health (CDC, 2015). In response to that threat, researchers have developed molecular assays to predict antimicrobial resistance (Melendez et al., 2018) based on the presence of mutations in antimicrobial target genes. Those assays may aid in individualizing treatment options for individuals with NG infection. Targeted antimicrobial treatment options may reduce the selection pressure on NG and slow down the continued emergence of resistant NG infections (Buono et al., 2012).

Investigators have genotyped the NG DNA gyrase subunit A (gyrA) gene, the target of fluoroquinolones like ciprofloxacin, and the penicillin-binding protein 2 (penA) gene, the target of third-generation cephalosporins like cefixime, in NG isolates as well as in clinical specimens from Hologic Aptima™ and Roche Cobas® NG detection assays (Donà et al., 2016; Hemarajata et al., 2016; Whiley et al., 2012; Wong et al., 2017). In order to expand the use of those assays to different testing platforms, we conducted a pilot study to determine if we could genotype the NG antimicrobial target genes gyrA and penA in remnant Cepheid CT/NG Xpert® clinical specimens (Wong et al., 2017).

We tested 61 frozen (−80° C), de-identified, remnant pharyngeal, rectal, urine, and vaginal NG-DNA positive specimens previously self-collected between May 2017 and January 2019 as part of the U.S. National Institutes of Health Adolescent Trials Network study (ATN) (Shannon et al., 2019). Specimens were tested with the Cepheid Xpert ® CT/NG Assay (Cepheid, Sunnyvale, CA), a qualitative real-time PCR assay, approved by the Food and Drug Administration for genital, pharyngeal, and rectal specimens.

Specimens were thawed at room temperature and 100μL of NG DNA was extracted from a total volume of 200μL from each specimen using the Roche® MagNA Pure System (Roche, Indianapolis, Indiana). We then used the Roche LightCycler 480 (Roche, Indianapolis, Indiana) to produce a high resolution, multiplex melt curve analysis for the detection of the gyrA codon serine 91 mutation and the penA mosaic XXXIV allele (Wong et al., 2017). The fluorescent labels of the detection probes for the penA mosaic XXXIV target (Cyanine-5 dye) differed from that of gyrA (LightCycler® 640 probe) so that both genes could be detected simultaneously at various wavelengths. For internal controls we used isolates with previously confirmed presence of the NG mutant gyrA, the NG wild type gyrA, and the mosaic penA XXXIV allele. Lastly, we presented the frequency of the gyrA serine 91 mutation and the penA XXIV allele by anatomic site: rectum, pharynx, urethra, and vagina.

Among the 61 NG positive clinical specimens, 52% (32/61) were successfully genotyped (Table): gyrA serine 91 mutant (16/32, 50%), wildtype (16/32, 50%), mosaic penA XXXIV (2/32, 6.3%), and non-mosaic penA (30/32, 94.7%). Overall, five out of six urine specimens (83%), 18/26 rectal samples (69%), 2/5 vaginal (40%) and 7/24 pharyngeal (30%) were genotyped. The table summarizes the frequency of penA and gyrA genotypes by anatomic site (rectal, pharyngeal, urethral, and vaginal).

Table:

Anatomical Distribution & Genotype of 32 Cepheid Xpert® remnant Neisseria gonorrhoeae specimens

Anatomical Site penA XXXIV absent (N=30) penA XXXIV present (N=2)
gyrA MT* (N=15) gyrA WT** (N=15) gyrA MT* (N=1) gyrA WT** (N=1)
n(%) n(%) n(%) n(%)
Rectal 9 (28.1) 8 (25.0) 1 (3.1) 0
Pharyngeal 3 (9.4) 3 (9.4) 0 1 (3.1)
Urethral 2 (6.3) 3 (9.4) 0 0
Vaginal 1 (3.1) 1 (3.1) 0 0
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**

WT= Wildtype

*

MT= Mutant

We tested remnant Cepheid Xpert® urogenital and extragenital specimens for the NG gyrA serine 91 mutation and mosaic penA XXXIV allele. In this pilot study, we showed that it is feasible to detect antimicrobial resistance genes in a small batch of remnant samples. We successfully genotyped about half of the previously collected and stored specimens that included both genital and extragenital samples. We show that genotyping of antimicrobial targets using remnant Cepheid Xpert ® clinical specimens is feasible, but additional improvements, particularly in pharyngeal specimens, are needed.

Of the successfully genotyped samples, half had a mutated gyrA gene predicting decreased susceptibility or resistance to ciprofloxacin. Another study found a similar prevalence of the gyrA resistance mutation among patients infected with NG (Bhatti et al., 2017). Genotyping the gyrA gene can help predict susceptibility to ciprofloxacin, which may allow for the use of ciprofloxacin for treatment and avoidance of last line antibiotics like ceftriaxone (Allan-Blitz et al., 2017).

Previous studies have shown that isolating NG genetic material from genital samples is more efficient than extragenital samples and there are differences in gyrA assay performance in different anatomical sites. In a recent publication from our team, gyrA was successfully genotyped in a higher proportion of urine/genital samples (76.4%), followed by rectal (67.2%) and pharyngeal samples (36.1%) (Ellis et al., 2019). In addition, another study tested 73 NG genital and extragenital samples and conducted sequencing of different gene targets (porB, tbpB) (Whiley et al., 2010). That study managed to successfully genotype all urogenital samples, 85% (11 of 13) of rectal samples and 57% (4 of 7) pharyngeal samples. Even though the latter group studied different genes, both studies showed high success in genotyping urogenital samples whereas extra-genital specimens were more difficult to genotype.

The bacterial load and presence of other Neisseria species might affect the performance of NG genotyping in clinical specimens. Whiley et al. (2010) observed cross-reaction between commensal, non-gonococcal Neisseria species in pharyngeal samples, which reduced the specificity of their assay. It is possible that saprophytic Neisseria species of the throat are cross-reacting and inhibiting the PCR reaction. An additional reason for a lower success rate of genotyping could be the differences in bacterial load in the different anatomical sites. Bacterial load may be lower in extragenital sites compared to genital, and even lower in pharyngeal compared to rectal (Bisessor et al., 2011).

Our study was limited by its modest sample size, which limits the generalizability of our findings and does not allow us to conduct comparisons on the assay performance between the different anatomical sites. In addition, NG isolates were not concurrently tested for phenotypic susceptibility, the gold standard for measuring antimicrobial susceptibility.

It is possible to use targeted genotyping of NG specimens as a novel means to strengthen antimicrobial stewardship in the rising threat of multi-drug resistant Neisseria gonorrhoeae. Our findings suggest that a variety of remnant specimen types may be used for successful genotyping of the antimicrobial target genes, however additional research is needed to improve detection performance. Large commercial CT/NG assay manufacturers should invest in the development of assays that predict antimicrobial susceptibility in NG infections in both genital and extragenital samples

Funding:

This work was supported in part by the National Institutes of Health awards 1U19HD089886 and 5R21AI117256.

Footnotes

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