Study gauges impact of genotyping on gonorrhea treatment

Amy Carpenter Aquino

April 2019—Genotypic testing for ciprofloxacin susceptibility in Neisseria gonorrhoeae has been proved to be effective in guiding physician treatment in a single-center study at UCLA Health.

Jeffrey D. Klausner, MD, MPH, a clinical professor in the Department of Medicine, Division of Infectious Diseases, and the Department of Epidemiology, David Geffen School of Medicine and the Fielding School of Public Health, University of California, shared the details of the study at last year’s Association for Molecular Pathology meeting and in a recent interview.

“The overall concept is that with rapid detection of Neisseria gonorrhoeae, and detection of key antimicrobial resistant genes, we can enable doctors to do targeted treatment, which will reduce antibiotic selection pressure and decrease the emergence of resistance,” Dr. Klausner said.

The British Association for Sexual Health and HIV endorsed this approach when it issued a gonorrhea guideline update in January, Dr. Klausner says. “They specifically recommend the use of molecular tests to predict susceptibility of antibiotics in Neisseria gonorrhoeae infections.” The new guideline removes the recommendation for dual therapy with azithromycin and encourages the use of ciprofloxacin in certain cases when a molecular test predicts susceptibility.

Dr. Klausner describes the “wake-up case” as that of a heterosexual man who presented to a sexual health clinic in the United Kingdom in December 2014 with a two-week history of urogenital symptoms (Fifer H, et al. N Engl J Med. 2016;374[25]:2504–2506). The patient had returned 10 days earlier from a trip to Japan, where his Japanese female partner had been treated for gonorrhea.

The patient’s nucleic acid amplification tests were positive for N. gonorrhoeae in a urine specimen and pharyngeal swab, and he had an N. gonorrhoeae-positive urethral culture. He received the standard dual treatment for N. gonorrhoeae infection in the U.K. at the time, which was a 500-mg injection of ceftriaxone plus one gram orally of azithromycin. (The Centers for Disease Control and Prevention currently recommends a similar dual therapy of a 250-mg injection of ceftriaxone with one gram orally of azithromycin.)

Phenotypic testing revealed that the patient’s N. gonorrhoeae strain was resistant to cefuroxime, ciprofloxacin, and tetracycline. The patient had follow-up pharyngeal NAATs on days 15, 79, and 98; all tested positive for N. gonorrhoeae. A culture taken on day 98 was also positive.

The patient was then treated with double doses of the initial treatment: a one-gram injection of ceftriaxone and two grams orally of azithromycin. “Finally, nearly four months later, his pharyngeal swab tested negative,” Dr. Klausner said.

Antimicrobial susceptibility testing by Etest of the pharyngeal isolate found resistance to ceftriaxone, cefixime, cefotaxime, azithromycin, penicillin, tetracycline, and ciprofloxacin. It was susceptible only to spectinomycin (which is not available in the United States).

Whole genome and conventional sequencing of the organism found multiple gene alterations. “They found what we call a mosaic penA gene, which has been classified as X,” Dr. Klausner said. “People have classified about three dozen different mosaic penA types associated with an altered penicillin-binding protein 2, with a decreased target affinity to ceftriaxone.” Other gene alterations were an mtrR promoter deletion of one adenine (which increases MtrCDE efflux of ceftriaxone and azithromycin) and a change in penB (which decreases the PorB influx of ceftriaxone and azithromycin).

Third-generation cephalosporin resistance in N. gonorrhoeae is rising in the U.S., as noted by CDC data published in the New England Journal of Medicine in 2012 (Bolan GA, et al. 366[6]:485–487). In 2011, Dr. Klausner said referring to the data, isolates from states in the West, from five percent of men who have sex with men, had higher levels of elevated MICs for decreased susceptibility to cefixime.

Drug-resistant N. gonorrhoeae has been declared one of the three most urgent infectious public health threats. “That’s a critical thing because it alerts funders and policymakers to this issue,” Dr. Klausner said. He was in Washington, DC, last summer, and “the one thing politicians knew about STDs,” he said, “was that there is untreatable gonorrhea out there,” which has resulted in more funding and research opportunities.

N. gonorrhoeae antibiotic resistance is a pattern that has persisted for decades, since the sulphonamides of the 1930s. But because the return on investment for a single-dose antibiotic for the infection has not been “particularly motivating” for drug developers, Dr. Klausner said, new strategies are needed. He has pursued one such strategy for the past decade.

In N. gonorrhoeae, there are multiple targets for different antimicrobials and multiple mechanisms of resistance. Dr. Klausner zeroes in on where fluoroquinolones work on the gyrA enzyme, which is coded by the gyrA gene. “As we develop better tools and understanding, we can learn more about how we can use the prediction from sequencing to look at antimicrobial susceptibility.”

N. gonorrhoeae is notorious for its promiscuity and ability to collect DNA elements, either plasmids or chromosomal genes from neighboring organisms, he said. “The oropharynx normally has a high level of Neisseria commensals. It’s normal in the throat to have a Neisseria cinerea, Neisseria subflava, other Neisseria species, and the thought is that by taking antibiotics, those other species may develop resistance.” When a patient acquires a gonococcal infection, there is easy transformation of those resistant elements to the new N. gonorrhoeae that have just landed in the throat, “a particularly good place for growth and the passage of resistant elements.”

CDC data from 2016 on antimicrobial susceptibility of N. gonorrhoeae isolates showed that 73 percent of isolates are ciprofloxacin susceptible, with 27 percent resistant. “I began thinking, we treat this bug with a sledgehammer by treating every infection with ceftriaxone plus azithromycin,” Dr. Klausner said. “If we actually knew the susceptibility profile of the organism at the time of treatment, we could probably be smarter. We could reduce the selection pressure on the organism by the use of different antibiotics.”

DNA gyrase A is the target of ciprofloxacin. “In a wild-type gyrase, the ciprofloxacin binds to that enzyme, inhibits the enzymatic activity, prevents a normal development of DNA, and the organism is nonviable,” Dr. Klausner said. In the mutated enzyme, the ciprofloxacin cannot bind or act and the organism is resistant. Different work has shown that a single point mutation at Ser-91 is associated with this mutation. “We’re lucky that one single point mutation was both necessary and sufficient,” he said. “While there are other mutations, such as in par C and gyrB, the singular presence of the alteration in Ser-91 can predict both sensitivity and resistance.”