Summary:
We determined the frequency of discordant gyrase A genotypes among concurrent but anatomically separate Neisseria gonorrhoeae infections.
Introduction
The emergence of multidrug-resistant Neisseria (N.) gonorrhoeae infections has caused great concern (1, 2), and has resulted in novel approaches to combat antimicrobial resistance (3). One approach utilizes antibiotics which are no longer recommended for empiric therapy for N. gonorrhoeae. That has been made possible by rapid molecular genotypic assays for predicting antimicrobial susceptibility (4). The gyrase A (gyrA) assay is a real-time molecular test which determines the presence of mutation at codon 91 of the gyrA gene (5); the absence of mutation at this locus has been shown to reliably predict susceptibility to ciprofloxacin (6). The use of ciprofloxacin as opposed to dual therapy with ceftriaxone and azithromycin affords several benefits including the reduction in selection pressure for the emergence of ceftriaxone resistance.
In November 2015, the University of California Los Angeles Health System implemented gyrA genotyping on all N. gonorrhoeae positive clinical specimens (7). In working with the gyrA assay, clinicians have questioned, among cases in which individuals have multiple anatomic sites involved, what is the likelihood that all infections have the same gyrA genotype? Previous studies have demonstrated that mixed infections with multiple strains of N. gonorrhoeae at the same anatomic site may occur (8, 9). Therefore, it is plausible that infections at different anatomic sites may also reflect different strains of N. gonorrhoeae, and therefore different antimicrobial susceptibility profiles. One study identified three cases of N. gonorrhoeae infections harboring strains with differing susceptibilities to ciprofloxacin at unique anatomic sites (10). Further data, however, are lacking. In order to test that hypothesis further, we aimed to characterize the frequency of discordant genotype results among cases that were successfully gyrA genotyped at the University of California Los Angeles Clinical Microbiology Laboratory and the Los Angeles Lesbian Gay Bisexual and Transgender Center and had more than one anatomic site of infection.
Methods
We performed a retrospective chart review of all N. gonorrhoeae cases with more than 1 anatomic site of involvement that were successfully genotyped between November 1st 2015 and April 28th 2017 at the University of California Los Angeles. Specimens that were N. gonorrhoeae positive as determined by the Cobas™ 4800 CT/NG Assay (Roche Molecular Systems, Pleasanton, CA, USA) were reflexed to gyrA genotyping. A single case was defined as a new diagnosis of N. gonorrhoeae infection at one or more anatomic site on a given date.
We also included N. gonorrhoeae positive specimens from participants presenting for routine sexual health screening at the Los Angeles Lesbian Gay Bisexual and Transgender Center between September 1st 2016 and February 28th 2018, tested at the Los Angeles County Public Health Laboratory (APTIMA® Combo 2 CT/NG assay; Gen-Probe, San Diego, CA, USA). Those specimens which were N. gonorrhoeae positive were also reflexed to gyrA genotyping.
GyrA genotyping was performed using a real-time polymerase chain reaction assay with high-resolution melt analysis. That, along with fluorescence resonance energy transfer probes, which target the codon 91 region of the gyrA gene, enabled us to determine wild-type (non-mutated) vs. mutant gyrA sequences. The sequences of the probes used were: 5’-GCA-TCG-TCG-GCG-ACG-TCA-TCG-GTA-AAT-ACC-ACC-C-3’-fluorescein and 5’-Red 640-ACG-GCG-ATT-CCG-CAG-TT-3’-phosphorylated.
We then determined the frequency of discordant genotype results and the 95% confidence interval. All analysis was performed using STATA software version 15.0 (StataCorp®, College Station, TX, USA). The study and the analysis were approved by the University of California Los Angeles institutional review board.
Results
During the study period 1,243 cases of N. gonorrhoeae infection were identified. Of those, 125 (10%) cases had greater than one anatomic site involved and were successfully genotyped. Four (3.2% [95% CI 0.9%−8.0%]) had discordant results (Table).
Table:
Four cases of Neisseria gonorrhoeae infection involving multiple anatomic sites with discordant gyrase A (gyrA) genotype results
| Case | Anatomic Site of Infection | GyrA Genotype |
|---|---|---|
| 1 | Urine | Mutant |
| Rectal | Wild-Type | |
| 2 | Urine | Mutant |
| Pharyngeal | Wild-Type | |
| 3 | Rectal | Mutant |
| Pharyngeal | Wild-Type | |
| 4 | Rectal | Mutant |
| Urine | Wild-Type | |
Mutant gyrA genotype infections are associated with ciprofloxacin resistance; Wild-type gyrA infections are associated with ciprofloxacin susceptibility.
Discussion
We determined the frequency of discordant gyrA genotype N. gonorrhoeae infections among patients with N. gonorrhoeae detected at more than one anatomic site, diagnosed at the University of California Los Angeles Clinical Microbiology Laboratory or at the Los Angeles Lesbian Gay Bisexual and Transgender Center. Our results provide evidence that a low proportion of anatomically separate N. gonorrhoeae infections in the same patient detected on the same date have discordant gyrA genotypes, and thus different susceptibilities to ciprofloxacin. That finding should provide reassurance in the routine introduction of gyrA testing into clinical practice and may obviate the need to perform genotyping on each anatomic site of infection in a single patient. However, should gyrA genotyping be done only from specimens collected from one anatomic site, there might be a small proportion of inadequately treated infections.
Our findings are in agreement with a prior study that identified a small, but notable proportion of cases of N. gonorrhoeae infections with multiple anatomic sites involved, which have different susceptibilities to ciprofloxacin and other antibiotics; that study found three cases (4.2%) where there were differences in ciprofloxacin susceptibility (10). Those three cases had infections at the rectum and urethra, while our results provided evidence that other combinations of anatomic sites may also be infected by different strains of N. gonorrhoeae.
Furthermore, our findings may provide the ground work for future studies investigating the relationship between anatomically isolated but concurrent infections and antimicrobial susceptibility. Such research may inform the transmission dynamics of resistance genes, and further inform implementation of rapid molecular assays.
Our study had several limitations. Primarily, the precision of our findings is limited by the sample size. Furthermore, given the methods of selecting cases, we were unable to determine the prevalence of discordant results. Finally, the samples were all collected in Los Angeles. Further studies should aim to include specimens from other regions. However, given our aim of characterizing the frequency of discordant results to test the hypothesis that N. gonorrhoeae from only one anatomic site needs to be gyrA genotyped among cases in which there are multiple anatomic sites infected, we feel that those limitations do not negate the importance of our findings.
In conclusion, the frequency of discordant gyrA genotype N. gonorrhoeae infections was low. Obtaining genotype results from one anatomic site may be sufficient to guide antimicrobial therapy. Further studies in larger samples and different populations are needed to identify the actual prevalence of discordant genotype results.
Acknowledgements:
This research was supported by the National Institutes of Health, grants: R21AI77256 and R21AI109005. The authors would also like to acknowledge Nicole Green and the NG GyrA Study group for their contributions to this project.
Footnotes
The authors have no conflicts of interests.
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