2022 STD Prevention Conference: Testing for Gonorrhea & Antimicrobial Resistance

introduction[00:00] Introduction      
Hello everyone. My name is Meena Ramchandani. I’m an infectious disease physician at the University of Washington in Seattle. This podcast is dedicated to an STD [sexually transmitted disease] literature review for health care professionals who are interested in remaining up-to-date on the diagnosis, management, and prevention of STDs.

background[00:21] Background

In this episode, we’ll review some oral abstracts presented at the STD Prevention Conference that was held virtually in September of 2022. I’d like to focus on the topic of testing for gonorrhea, including antimicrobial resistance testing. The session we are going to focus on was titled “Molecular Detection of Neisseria gonorrhoeae Antimicrobial Resistance and STI Strain Characterization,â€? and it was presented on September 19, 2022. 

So why is this important? Antimicrobial-resistant gonorrhea is an urgent public health threat, especially since many circulating isolates have developed resistance to several different classes of antibiotics. Now, typically, antimicrobial susceptibility testing requires culture of an isolate, but now that NAATs, or nucleic acid amplification tests, are the primary way we diagnose gonorrhea, culture samples are rarely obtained in clinical practice. Also, culture samples take more time for results to return. One question that comes up is, is there a way to detect antimicrobial resistance on NAAT samples that are positive for gonorrhea? Different groups are evaluating ways to assess for antimicrobial resistance using NAAT specimens.

oral-session-11[01:36] Oral Session 1.1

The first session was presented by Shelby Hutton, a fellow in the Division of STD Prevention at the CDC. Her group evaluated molecular assays for the detection of antimicrobial resistance-associated mutations in Neisseria gonorrhoeae from clinical NAAT specimens. 

1. Now this group they have developed assays to target seven genetic mutations that are associated with three different antimicrobial resistance determinants to different antibiotic classes. This includes the extended-spectrum cephalosporins (such as like, for example, ceftriaxone or cefixime), the fluoroquinolones (such as ciprofloxacin), as well as the macrolides (or azithromycin).
2. Using RT PCR—or reverse transcription PCR—they can detect these mutations in NAAT samples using wild-type as well mutant probes, which compete for binding to the template DNA.
3. Now, first, they ran these assays for antimicrobial resistance on a diverse panel of 88 isolates, and then they compared it to the whole-genome sequencing data for each of these isolates.
4. The authors found that the majority of isolates were correctly called for each of the mutations when this antimicrobial resistance assay was compared to whole-genome sequencing for both mutant and wild-type isolates. They only had less than three failures occur when there was a discrepancy in the mutations detected by the assay compared to whole-genome sequencing. 
5. The assay they developed was then run on 32 NAAT specimens with paired culture isolates and compared to whole-genome sequencing data, and what they found is that the majority of NAAT specimens were correctly called for the different antimicrobial resistance determinants that they have thus far evaluated, which was five in total.

So this was a great presentation, and the reason I liked it is because they were discussing a molecular assay to detect markers of antimicrobial resistance for Neisseria gonorrhoeae. Overall, it sounds like the majority of isolates were correctly called when they compared this information of the assay to whole-genome sequencing. The benefits of this type of assay could include enhancing gonorrhea antimicrobial resistance surveillance but also informing individualized gonorrhea treatment. For example, let’s say you have a patient in clinic; being able to identify which antibiotics could be used to treat their infection would help to reduce unnecessary antibiotics but also help to ensure the correct treatment was more immediately prescribed. The group did notice a few failures to detect mutations associated with drug resistance on NAAT specimens. And what they found is that the failure to detect mutations were due to low concentration of gonorrhea DNA in the NAAT specimens, so just something to consider for future study.

oral-session-12[04:21] Oral Session 1.2 

The second study session was presented by Dr. Olusegun Soge, an Assistant Professor at the University of Washington, where he and his group evaluated the performance of ResistancePlus GC Assay for detection of ciprofloxacin resistance in Neisseria gonorrhoeae. 

1. ResistancePlus GC Assay is a commercial assay for simultaneous detection of gonorrhea and either resistance or susceptibility to ciprofloxacin. So the way this assay works is by having two targets for detection of the organism, and then a reflex assay is performed to detect whether a mutant gyrA gene is present, indicating antimicrobial resistance to ciprofloxacin. More specifically, the gyrA S91F mutation is highly predictive of gonorrhea ciprofloxacin resistance. 
2. What the authors found is that the 14 isolates and 164 remnant Aptima specimens with known gyrA S91F mutation they analyzed had 100% agreement with expected gyrA genotype and ciprofloxacin MICs.
3. Now, since some limitations of these types of molecular assays would include possible genomic cross-reactivity with, let’s say, commensal Neisseria species, they analyzed this a bit further. And what they found is that no cross-reactivity was observed in 40 commensal Neisseria species and 100 pharyngeal, rectal, urine, and vaginal specimens that were gonorrhea-negative by Aptima.
4. When they looked at 200 archived previously frozen Aptima specimens with paired gonorrhea isolates, they found 90% concordance with ResistancePlus GC Assay.  
5. Overall, the ResistancePlus GC Assay showed a sensitivity of 99% and a specificity of 98% using the 180 Aptima specimens paired with gonorrhea isolates with ciprofloxacin MICs. 

Now right now, the ResistancePlus GC assay is not FDA-approved and needs rigorous lab validation. In this study, ResistancePlus GC Assay had good performance characteristics for Neisseria gonorrhoeae. The authors found that indeterminate gyrA results did occur and were more likely to occur in frozen specimens and recent extragenital specimens. Now, this might be due to low DNA yield from frozen samples or lower sensitivity of the ResistancePlus assay for gonorrhea compared to the Aptima test, but further studies are needed in this area. It’s exciting to think of these types of assays are being studied as they can really inform clinical decision-making, enhance surveillance, as well as overall public health response for Neisseria gonorrhoeae isolates in the U.S. 

oral-session-13[07:05] Oral Session 1.3

The next abstract was presented by Teresa Abraham, who’s a vice president at Visby Medical. She discussed using a portable palm-sized PCR platform, which is called the Visby Medical Sexual Health, to help with the diagnosis of gonorrhea as well as antimicrobial resistance testing as a point-of-care test. 

1. The assay takes less than 30 minutes to perform and, per the authors, is easy to use and interpret. So, what the assay does, is it looks for a single nucleotide polymorphism—also known as a SNP—that has been shown to predict ciprofloxacin susceptibility in gonorrhea. This SNP is located on the gyrA gene: the same gyrA gene as I mentioned in the last presentation.
2. Now, from a single vaginal swab sample, which is self-collected, the assay detects whether the sample has gonorrhea and then determines if the infecting gonorrhea strain is susceptible to ciprofloxacin. It’s designed to use either as a standalone test or as a reflex test after the initial diagnosis of gonorrhea infection has been made. 
3. To evaluate further, the authors tested 11 samples on this assay, and they found that nine samples were susceptible to ciprofloxacin. Six out of the nine samples were then sent for whole-genome sequencing and found to have the ciprofloxacin susceptibility SNP, indicating that the assay correctly called ciprofloxacin susceptibility. Two samples were found to be resistant to ciprofloxacin, and whole-genome sequencing for both these strains found that they do, in fact, have the mutations conferring ciprofloxacin resistance. 

Now right now, this assay is not commercially available, and this is a very small sample size, but it’s great to think of diagnosing gonorrhea in the clinical setting in a rapid way and possibly helping to provide susceptibility-guided therapy in the moment as a point-of-care test. This can also possibly preserve the efficacy of ceftriaxone, one of the last medications used to treat gonorrhea that is currently available without resistance-guided therapy. The authors mention in the future, this test might be used as a prototype for a molecular point-of-care test, either as a standalone test, or for antibiotic susceptibility reflex testing where follow-up cannot be ensured.

oral-session-14[09:17] Oral Session 1.4

The last oral abstract I want to cover is a bit different, but I wanted to include it in this episode. It was presented by Dr. Mathew Schmerer in the Division of STD Prevention at the CDC. 

1. The group evaluated ways to improve cluster analysis for gonorrhea, and this is important for surveillance as well as public health, using whole-genome sequencing data.
2. One way to evaluate clusters is to use SNPs (single nucleotide polymorphisms) and look for changes in alignment. But there might be other ways for cluster analysis. For example, those that might be easier or more convenient. Another way for cluster analysis is to use multilocus sequence typing, and this looks at various genes and is called MLST. 
3. Two different variations of using MLST are available. One includes core genome MLST, which covers about 1600 genes. Another variation includes whole-genome MLST, which covers about 2400 genes.
4. Now, there are advantages and disadvantages to each type of evaluation, which I won’t get into the details in this episode. But this group decided to explore MLST data as the basis for cluster analysis for gonorrhea.
5. They evaluated 278 gonorrhea sequences from one SURRG site and built both a core genome and a whole-genome MLST scheme—now, SURRG stands for Strengthening the United States Response to Resistant Gonorrhea, and it began in 2016. It’s a program to enhance local antibiotic-resistant gonorrhea preparedness capacity. 
6. The authors found that both frameworks (both the core genome MLST as well as the whole-genome MLST) produced similar clustering of isolates. So when evaluating cluster analysis, it seems as though the core genome MLST, which is looking at a set of about 1600 genes, can be a candidate for future exploration of cluster analysis Neisseria gonorrhoeae. 

Overall, this was a great session that explored an important area of research for gonorrhea testing. It included reviewing the use of molecular assays to detect markers of antimicrobial resistance or novel methods to evaluate clusters for surveillance purposes. Gonorrhea has progressively developed resistance to antibiotics typically used to treat this pathogen, and we have very few antibiotic options left. This makes it really challenging in the clinical setting. It’s important to monitor resistance, and by using these types of techniques, I could see how these assays can help clinicians in the future with more rapid ways to treat each case of gonorrhea quickly and effectively with the right antibiotic, even potentially in real-time. I look forward to hearing more about these assays and their use in the clinical setting in the future. 

summary[12:04] Summary

To conclude, I’d like to summarize some key points from this session:

1. Antimicrobial-resistant gonorrhea is an urgent public health threat, and using molecular assays to detect markers of antimicrobial resistance might be helpful in the future to enhance surveillance and possibly inform individualized gonorrhea treatment. There are some assays in research settings that are currently being explored. 
2. Antimicrobial resistance determinants that have been identified have the potential to evaluate mutations associated with resistance to various antibiotics such as cephalosporins, fluoroquinolones, and/or macrolides.
3. A palm-sized PCR platform for the diagnosis of gonorrhea as well as antimicrobial resistance testing is being investigated as a point-of-care test on self-collected vaginal swabs.
4. Cluster analysis of gonorrhea is an important area of surveillance and core genome MLST might be a candidate for future exploration.

credits[13:05] Credits      
This podcast is brought to you by the National STD Curriculum, the University of Washington STD Prevention Training Center, and is funded by the Centers for Disease Control and Prevention. 

Transcripts and references for this podcast series can be found on our website, the National STD Curriculum, at www.std.uw.edu. Thank you for listening, and have a wonderful day.