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Centers for Disease Control and Prevention (CDC)

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Drug-Resistant Gonorrhea: An Urgent Public Health Issue

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What is Gonorrhoea? | Why is Untreated Gonorrhoea so Serious?

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Join Dr. Rowley as he talks over some of the most commonly asked questions about gonorrhoea infection. See below for an overview of points covered in this video. For more information please navigate to: https://www.letsgetchecked.com/gb/en/sexual-health/sti-testing/?utm_source=youtube&utm_medium=video&utm_campaign=Rowley_Videos_YT&utm_term=Gonorrhoea%20YT%20video%202&utm_content=video%20description 00:18 - What is gonorrhoea? 00:37 - How is gonorrhoea spread?

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Recommendations

Based on the following information, the American College of Obstetricians and Gynecologists makes these recommendations:

Dual therapy with ceftriaxone and azithromycin should be administered. It should be administered together on the same day, preferably simultaneously, and under direct observation.

Test-of-cure is not recommended for women diagnosed with uncomplicated urogenital or rectal gonorrhea treated with the recommended or alternative regimens.

Pregnant women treated with dual therapy for gonorrhea do not require a test-of-cure.

Women with pharyngeal gonorrhea treated with an alternative regimen should return 14 days after treatment for a test-of-cure using either culture or nucleic acid amplification test (NAAT).

The Centers for Disease Control and Prevention's (CDC) guidance (www.cdc.gov/mmwr/preview/mmwrhtml/rr6403a1.htm?s_cid=rr6403a1_e) on the treatment of gonococcal infections is likely to evolve in the coming years, and obstetrician-gynecologists should remain aware of new developments in this area. The CDC web site (www.cdc.gov/std/gisp/) and state health departments can provide the most current information on gonococcal susceptibility.

Gonorrhea is the second most commonly reported bacterial sexually transmitted disease (STD) in the United States, with an estimated 820,000 new Neisseria gonorrhoeae infections occurring each year ( 1). Antimicrobial resistance limits treatment success, heightens the risk of complications, and may facilitate the transmission of sexually transmitted infections (STIs). Neisseria gonorrhoeae has developed resistance to sulfonamides, tetracyclines, and penicillin. In 2007, emergence of fluoroquinolone-resistant N gonorrhoeae prompted the CDC to no longer recommend fluoroquinolones for treatment of gonorrhea, leaving cephalosporins as the only remaining class of recommended antimicrobials ( 2).

In an attempt to prevent development of resistance to cephalosporins, the CDC's 2010 STD treatment guidelines recommended combination therapy for gonorrhea with a cephalosporin plus either azithromycin or doxycycline, even if NAAT for Chlamydia trachomatis was negative at the time of treatment ( 3). This emerging cephalosporin resistance was manifested from 2006 to 2011, when the minimum inhibitory concentrations of cefixime needed to inhibit the growth in vitro of N gonorrhoeae strains in the United States increased, suggesting that the effectiveness of cefixime might be waning ( 4, 5). As a result, cefixime is no longer a first-line regimen. Dual therapy with ceftriaxone and azithromycin remains the only recommended first-line regimen for the treatment of gonorrhea in the United States (1, 5).

Treatment Regimens

Dual therapy with ceftriaxone and azithromycin should be administered together on the same day, preferably simultaneously, and under direct observation (see Box 1). Ceftriaxone in a single-intramuscular (IM) injection of 250 mg provides sustained, high bactericidal levels in the blood; clinical experience indicates that ceftriaxone is safe and effective for the treatment of uncomplicated gonorrhea at all anatomic sites, curing more than 98% of all infections ( 6, 7). The use of azithromycin as the second antimicrobial is preferred to doxycycline because of the convenience and adherence advantages of single-dose therapy and the substantially higher prevalence of gonococcal resistance to tetracycline than to azithromycin (1, 8). The use of ceftriaxone or cefixime is contraindicated in those patients with a history of an IgE mediated β-lactam allergy, such as anaphylaxis, Stevens-Johnson syndrome, or toxic epidermal necrolysis (see Box 1 for alternate treatment regimens) ( 9, 10).

Recent clinical trials showed that dual treatment with a single 320-mg dose of oral gemifloxacin plus a 2-g dose of oral azithromycin, or dual treatment with a single 240-mg dose of IM gentamicin plus a 2-g dose of oral azithromycin, were effective for uncomplicated urogenital gonorrhea ( 11). Either of these regimens may be considered as alternative treatment options when ceftriaxone is contraindicated. Monotherapy with a 2-g dose of oral azithromycin as a single dose in women who are not pregnant is no longer recommended in the United States ( 12-16).

Pregnancy

Pregnant women who are infected with N gonorrhoeae should be treated with the recommended dual therapy. A test-of-cure is not needed for individuals diagnosed with uncomplicated urogenital or rectal gonorrhea who are treated with the recommended or alternative regimen. Patients with severe penicillin or cephalosporin allergy can be administered dual therapy with a 240-mg dose of IM gentamicin and 2-g oral azithromycin. The use of gentamicin to treat chorioamnionitis demonstrates its safety in pregnancy ( 17, 18). Based on expert opinion, another alternative is a 2-g single dose of oral azithromycin in patients who are allergic to gentamicin and cephalosporins, but this category of patients need a test-of-cure 1 week after treatment. Such patients also may be referred to an infectious disease specialist. Dual therapy with ceftriaxone and azithromycin is recommended if the patient has a history of rash without anaphylaxis manifestations. Neither doxycycline nor quinolones are recommended during pregnancy.

Human Immunodeficiency Virus Infection

Patients infected with human immunodeficiency virus (HIV) with gonococcal infection should receive the same recommended dual therapy as those who do not have HIV (1). See general treatment recommendations described under "Treatment Regimens."

Management of Sex Partners

Recent sex partners within 60 days of diagnosis should be encouraged to seek evaluation and presumptive treatment for N gonorrhoeae and C trachomatis infections. If the last potential exposure was greater than 60 days, the most recent sex partner should be treated. Patients and sex partners should abstain from sexual activity for 7 days after treatment and until sex partners are adequately treated. If an obstetrician-gynecologist or other health care provider cannot ensure that an infected patient's partner will be promptly linked to care, and where legally accepted, expedited partner therapy with cefixime (400 mg) and azithromycin (1 g) should be delivered to the partner by the patient, a disease investigation specialist, or through a collaborating pharmacy. The CDC maintains a web site with information about the legal status of expedited partner therapy in all 50 states and other jurisdictions ( 19); additional information is available from the Guttmacher Institute ( 20). More information on expedited partner therapy is available in the American College of Obstetricians and Gynecologists' Committee Opinion Number 632, Expedited Partner Therapy in the Management of Gonorrhea and Chlamydial Infection ( 21). Expedited partner therapy should be accompanied by written materials educating partners about their exposure to gonorrhea, the importance of therapy, and when to seek clinical evaluation for reactions or complications.

Retesting

Appropriately treated patients do not need a test-of-cure. Retesting to detect repeat infection is distinct from test-of-cure to detect therapeutic failure. For patients who get retested and receive positive test results, most are more likely to be from reinfection than from treatment failure. Such patients should be retreated with the recommended dual regimen (250 mg of ceftriaxone IM plus 1 g of azithromycin orally). Patients who have symptoms that persist after treatment should be evaluated by culture for N gonorrhoeae (preferably with simultaneous NAAT), and any gonococci isolated should be tested for antimicrobial susceptibility. Persistent urethritis, cervicitis, or proctitis also might be caused by C trachomatis or other organisms.

Suspected Cephalosporin Treatment Failure

Cephalosporin treatment failure is defined as the persistence of N gonorrhoeae infection despite appropriate cephalosporin treatment and is indicative of infection with cephalosporin-resistant gonorrhea in individuals whose partners were adequately treated and whose risk of reinfection is low. Suspected treatment failure has been reported among individuals receiving oral and injectable cephalosporins ( 22, 23). Treatment failure should be considered in individuals whose symptoms do not resolve within 3-5 days after appropriate treatment and who report no sexual contact during the posttreatment follow-up period, and in individuals with a positive test-of-cure (eg, a positive culture after more than 72 hours or a positive NAAT result 7 or more days after receiving recommended treatment) and who report no sexual contact during the posttreatment follow-up period. Treatment failure also should be considered in individuals who have a positive culture on test-of-cure (if obtained) and there is evidence of decreased susceptibility to cephalosporins on antimicrobial susceptibility testing, regardless of whether sexual contact is reported during the posttreatment follow-up period.

In cases where reinfection is unlikely and treatment failure is suspected, before retreatment of the individual, relevant clinical specimens should be obtained for culture (preferably with simultaneous NAAT) and antimicrobial susceptibility testing should be performed if N gonorrhoeae is isolated. All isolates should be sent to the CDC for antimicrobial susceptibility testing; local laboratories should store isolates for possible further testing if needed. Instructions for shipping isolates to CDC can be found at www.cdc.gov/std/gonorrhea/arg/specimen_shipping_instructions1-29-08.pdf ( 24).

For individuals with suspected cephalosporin treatment failure, the treating clinician should consult an infectious disease specialist, an STD/HIV Prevention Training Center clinical expert ( www.nnptc.org) ( 25), the local or state health department STD program, or the CDC for advice. Dual treatment with a single 320-mg dose of oral gemifloxacin plus a 2-g dose of oral azithromycin or dual treatment with a single 240-mg dose of gentamicin IM plus a 2-g dose of oral azithromycin may be considered, particularly when isolates are found to have elevated cephalosporin minimum inhibitory concentrations ( 26). Individuals with suspected treatment failure after treatment with the alternative regimen (cefixime and azithromycin) should be treated with a single 250-mg IM dose of ceftriaxone and a single 2-g oral dose of azithromycin. A test-of-cure at relevant clinical sites should be obtained 7-14 days after retreatment; culture is the recommended test, preferably with simultaneous NAAT and antimicrobial susceptibility testing of N gonorrhoeae if isolated. Obstetrician-gynecologists or other health care providers should encourage the patient's sex partners from the preceding 60 days to seek prompt evaluation with culture and presumptive treatment using the same regimen used for the patient.

Follow-Up

Repeat N gonorrhoeae infection is prevalent among patients who have been diagnosed with and treated for gonorrhea in the preceding several months ( 27-29). Most of these infections result from reinfection and clinicians should therefore advise patients with gonorrhea to be retested 3 months after treatment. If patients do not seek medical care for retesting in 3 months, obstetrician-gynecologists or other health care providers are encouraged to test these patients whenever they next seek medical care within the following 12 months, regardless of whether the patients believe that their sex partners were treated. Pregnant women with antenatal gonococcal infection should be retested in the third trimester unless recently treated.

References

1. Cephalosporin susceptibility among Neisseria gonorrhoeae isolates-United States, 2000-2010. Centers for Disease Control and Prevention (CDC). MMWR Morb Mortal Wkly Rep 2011;60:873-7. [PubMed] [Full Text] ⇦

2. Waters LJ, Boag FC, Betournay R. Efficacy of azithromycin 1 g single dose in the management of uncomplicated gonorrhoea. Int J STD AIDS 2005;16:84. [PubMed] [Full Text] ⇦

3. Locksmith GJ, Chin A, Vu T, Shattuck KE, Hankins GD. High compared with standard gentamicin dosing for chorioamnionitis: a comparison of maternal and fetal serum drug levels. Obstet Gynecol 2005;105:473-9. [PubMed] [Obstetrics & Gynecology] ⇦

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Based on the following information, the American College of Obstetricians and Gynecologists makes these recommendations: Dual therapy with ceftriaxone and azithromycin should be administered. It should be administered together on the same day, preferably simultaneously, and under direct observation. Test-of-cure is not recommended for women diagnosed with uncomplicated urogenital or rectal gonorrhea treated with the recommended or alternative regimens.

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by Malak Sadek and Rana Elyamany
Introduction
Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea which is is the second most prevalent bacterial sexually transmitted infection worldwide. Its formal identification was in 1879 by the German bacteriologist Albert Neisser. Gonorrhea grows mainly in the warm, moist areas of the reproductive tract for both men and women. It can also grow in the mouth, throat, eyes, and anus.
Disease
Neisseria gonorrhoeae infections are acquired in humans by sexual contact. It is able to infect the lower genital tract, urethra in men and cervix in women (see Figure 1 and 2). Infected women may be asymptomatic (show no symptoms), but up to 50% show nonspecific symptoms including odorless mucopurulent, vaginal discharge and vaginal bleeding. Even infections without symptoms can also result in severe consequences. On the other hand, 90% of men with urethral infection have symptomatic mucopurulent penile discharge and dysuria. Gonococci can ascend to the upper genital tract, leading to serious diseases, such as epididymitis in men and cervicitis, endometriosis, and pelvic inflammatory disease in women. When N. gonorrhoeae infects the urogenital tract, it interacts with a variety of cells, including Polymorphonuclear leukocytes (PMNs or neutrophils). PMNs are professional cells of the immune system which often serve as the first line of host defense against the bacterial infection. N. gonorrhoeae can survive and replicate inside PMNs and this interactions likely play a critical role in the pathogenesis of infection (see Figure 3).
Epidemiology
Gonorrhea is a very common infectious disease. It is the second most prevalent sexually acquired infection in the United States, with more than 300,000 reported cases per year. The Center for Disease Control and Prevention (CDC) estimates that annually more than 700,000 people in the United States get new gonorrhea infections. The most recent data provided by the (CDC) indicate that reported cases have increased by almost 10% over the last 5 years. In 2013, 106.1 cases of N. gonorrhoeae per 100,000 persons were reported, representing an 8.2% increase in incidence from 2009. Approximately 75 percent of all reported cases of gonorrhea are found in younger persons 15 to 29 years of age. The prevalence of this infection has strong economic effects, as in 2008, the total lifetime direct medical cost of N. gonorrhoeae infections in the United States was estimated to be $162.1 million.
Virulence systems
Neisseria gonorrheae express a set of common mechanisms that allow its adaptation to the immune system and immune evasion. . Gonorrheae first attaches itself to the cells and the tissues using a rod shaped protein structure called Type IV pili that recognizes a molecule of the host and bind to it (the host is the human in whom the bacteria live and cause infection). Therefore Type IV pili is essential for virulence in N. gonorrhoeae because it mediates specific attachment to human mucosal cells, initiating the infectious process. It is known that type IV pili is formed from subunits of a protein called pilin, and a gene called pilE in the genome of N. gonorrhoeae, is responsible for encoding the pilin subunit. Notably, Type IV pili in gonorrhoeae is a surface antigen, an antigen is a substance that causes the immune system to produce large proteins called antibodies to identify and neutralize the bacteria. Because N. gonorrhoeae undergoes antigenic variation, the capacity to generate much more diversity in its surface antigens, it has the ability to resist the killing cells in the host. A mechanism called gene shuffling or gene conversion allows N. gonorrhoeae undergoes antigenic variation events.Through DNA recombination (DNA exchange between genes),pilE gene can exchange a part of itself with pilS gene copy. Subsequently a new variant of pilE protein will be produced which is not the same as the original one (see Figure 4). Producing different types of pili increases the chance of evading the immune system especially the antibodies response. Antibodies should bind to adhesions such as typeIV pili and prevent the attachment of the bacteria to the host cells. Furthermore antibodies increase the attachment of the bacteria to phagocytes (immune system cells that pick up and kill the bacteria by a process called phagoctosis) .Therefore, it is assumed that antibodies reacting with pili type IV could block the infection. However, switching of pili expression states alters bacterial interactions with host cells. Thus varying the antigens that are presented to the host immune system prevents antibodies binding to the bacterial surface and subsequently phagocytosis.
Treatment
N. gonorrhoeae has developed resistance to mainly all antibiotics introduced for treatment of gonorrhea. These drugs include: cefixime (an oral cephalosporin), ceftriaxone (an injectable cephalosporin), azithromycin, and tetracycline. N. gonorrhoeae is able to use number of mechanisms for antibiotics resistance including enzymes to degrade antibiotics. Recently, Centers for Disease Control and Prevention (CDC) recommends only ceftriaxone plus either azithromycin or doxycycline as first-line treatment for gonorrhea.
References:
Hill, S. A., & Davies, J. K. (2009). Pilin gene variation in Neisseria gonorrhoeae: reassessing the old paradigms. FEMS Microbiology Reviews, 33(3), 521-530.

JANET A. M. FYFE, C.S.C., AND JOHN K. DAVIES. 1995. The pilE Gene of Neisseria gonorrhoeae MS11 Is Transcribed from a s70 Promoter during Growth In Vitro. JOURNAL OF BACTERIOLOGY 177, No. 13: 3781-3787.

Lancaster et al (2015) Update on Treatment Options for Gonococcal Infections. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy.

Rotman and seifet (2014) The Genetics of Neisseria Species. Annual Reviews 48: 405-431. doi: 10.1146/annurev-genet-120213-092007.

Seifert, A.K.C.a.H.S. 2012. A bacterial siren song: intimate interactions between Neisseria and neutrophils. Nature Reviews Microbiology 10, 178-190. doi: 10.1038/nrmicro2713.

Unemo, M., & Shafer, W. M. (2011). Antibiotic resistance in Neisseria gonorrhoeae: origin, evolution, and lessons learned for the future. Annals of the New York Academy of Sciences, 1230, E19-E28. http://doi.org/10.1111/j.1749-6632.2011.06215.x

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by Malak Sadek and Rana Elyamany Introduction Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea which is is the second most prevalent bacterial sexually transmitted infection worldwide. Its formal identification was in 1879 by the German bacteriologist Albert Neisser. Gonorrhea grows mainly in the warm, moist areas of the reproductive tract...

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What is gonorrhea?

Gonorrhea is a sexually transmitted disease (STD) caused by infection with the Neisseria gonorrhoeae bacterium. N. gonorrhoeae infects the mucous membranes of the reproductive tract, including the cervix, uterus, and fallopian tubes in women, and the urethra in women and men. N. gonorrhoeae can also infect the mucous membranes of the mouth, throat, eyes, and rectum.

How common is gonorrhea?

Gonorrhea is a very common infectious disease. CDC estimates that approximately 1.6 million new gonococcal infections occurred in the United States in 2018, and more than half occur among young people aged 15-24. Gonorrhea is the second most commonly reported bacterial sexually transmitted infection in the United States. 2 However, many infections are asymptomatic, so reported cases only capture a fraction of the true burden.

How do people get gonorrhea?

Gonorrhea is transmitted through sexual contact with the penis, vagina, mouth, or anus of an infected partner. Ejaculation does not have to occur for gonorrhea to be transmitted or acquired. Gonorrhea can also be spread perinatally from mother to baby during childbirth.

People who have had gonorrhea and received treatment may be reinfected if they have sexual contact with a person infected with gonorrhea.

Who is at risk for gonorrhea?

Any sexually active person can be infected with gonorrhea. In the United States, the highest reported rates of infection are among sexually active teenagers, young adults, and African Americans .

What are the signs and symptoms of gonorrhea?

Many men with gonorrhea are asymptomatic . When present, signs and symptoms of urethral infection in men include dysuria or a white, yellow, or green urethral discharge that usually appears one to fourteen days after infection . In cases where urethral infection is complicated by epididymitis, men with gonorrhea may also complain of testicular or scrotal pain.

Most women with gonorrhea are asymptomatic . Even when a woman has symptoms, they are often so mild and nonspecific that they are mistaken for a bladder or vaginal infection . The initial symptoms and signs in women include dysuria, increased vaginal discharge, or vaginal bleeding between periods. Women with gonorrhea are at risk of developing serious complications from the infection, regardless of the presence or severity of symptoms.

Symptoms of rectal infection in both men and women may include discharge, anal itching, soreness, bleeding, or painful bowel movements . Rectal infection also may be asymptomatic. Pharyngeal infection may cause a sore throat, but usually is asymptomatic .

What are the complications of gonorrhea?

Untreated gonorrhea can cause serious and permanent health problems in both women and men.

In women, gonorrhea can spread into the uterus or fallopian tubes and cause pelvic inflammatory disease (PID). The symptoms may be quite mild or can be very severe and can include abdominal pain and fever . PID can lead to internal abscesses and chronic pelvic pain. PID can also damage the fallopian tubes enough to cause infertility or increase the risk of ectopic pregnancy.

In men, gonorrhea may be complicated by epididymitis. In rare cases, this may lead to infertility .

If left untreated, gonorrhea can also spread to the blood and cause disseminated gonococcal infection (DGI). DGI is usually characterized by arthritis, tenosynovitis, and/or dermatitis . This condition can be life threatening.

What about gonorrhea and HIV?

Untreated gonorrhea can increase a person's risk of acquiring or transmitting HIV, the virus that causes AIDS .

How does gonorrhea affect a pregnant woman and her baby?

If a pregnant woman has gonorrhea, she may give the infection to her baby as the baby passes through the birth canal during delivery. This can cause blindness, joint infection, or a life-threatening blood infection in the baby . Treatment of gonorrhea as soon as it is detected in pregnant women will reduce the risk of these complications. Pregnant women should consult a health care provider for appropriate examination, testing, and treatment, as necessary.

Who should be tested for gonorrhea?

Any sexually active person can be infected with gonorrhea. Anyone with genital symptoms such as discharge, burning during urination, unusual sores, or rash should stop having sex and see a health care provider immediately.

Also, anyone with an oral, anal, or vaginal sex partner who has been recently diagnosed with an STD should see a health care provider for evaluation.

Some people should be tested (screened) for gonorrhea even if they do not have symptoms or know of a sex partner who has gonorrhea . Anyone who is sexually active should discuss his or her risk factors with a health care provider and ask whether he or she should be tested for gonorrhea or other STDs.

CDC recommends yearly gonorrhea screening for all sexually active women younger than 25 years, as well as older women with risk factors such as new or multiple sex partners, or a sex partner who has a sexually transmitted infection.

People who have gonorrhea should also be tested for other STDs.

How is gonorrhea diagnosed?

Urogenital gonorrhea can be diagnosed by testing urine, urethral (for men), or endocervical or vaginal (for women) specimens using nucleic acid amplification testing (NAAT) . It can also be diagnosed using gonorrhea culture, which requires endocervical or urethral swab specimens.

FDA-cleared rectal and oral diagnostic tests for gonorrhea (as well as chlamydia) have been validated for clinical use .

What is the treatment for gonorrhea?

Gonorrhea can be cured with the right treatment. CDC now recommends a single 500 mg intramuscular dose of ceftriaxone for the treatment of gonorrhea. Alternative regimens are available when ceftriaxone cannot be used to treat urogenital or rectal gonorrhea. Although medication will stop the infection, it will not repair any permanent damage done by the disease. Antimicrobial resistance in gonorrhea is of increasing concern, and successful treatment of gonorrhea is becoming more difficult . A test-of-cure - follow-up testing to be sure the infection was treated successfully - is not needed for genital and rectal infections; however, if a person's symptoms continue for more than a few days after receiving treatment, he or she should return to a health care provider to be reevaluated. A test-of-cure is needed 7-14 days after treatment for people who are treated for pharyngeal (infection of the throat) gonorrhea.

Because re-infection is common, men and women with gonorrhea should be retested three months after treatment of the initial infection, regardless of whether they believe that their sex partners were successfully treated.

What about partners?

If a person has been diagnosed and treated for gonorrhea, he or she should tell all recent anal, vaginal, or oral sex partners so they can see a health provider and be treated . This will reduce the risk that the sex partners will develop serious complications from gonorrhea and will also reduce the person's risk of becoming reinfected. A person with gonorrhea and all of his or her sex partners must avoid having sex until they have completed their treatment for gonorrhea and until they no longer have symptoms. For tips on talking to partners about sex and STD testing, visit http://www.gytnow.org/talking-to-your-partner.

How can gonorrhea be prevented?

Latex condoms, when used consistently and correctly, can reduce the risk of transmission of gonorrhea . The surest way to avoid transmission of gonorrhea or other STDs is to abstain from vaginal, anal, and oral sex, or to be in a long-term mutually monogamous relationship with a partner who has been tested and is known to be uninfected.

Sources

1. Kreisel KM, Spicknall IH, Gargano JW, Lewis FM, Lewis RM, Markowitz LE, Roberts H, Satcher Johnson A, Song R, St. Cyr SB, Weston EJ, Torrone EA, Weinstock HS. Sexually transmitted infections among US women and men: Prevalence and incidence estimates, 2018. Sex Transm Dis 2021; in press.

2. CDC. Sexually Transmitted Disease Surveillance, 2021. Atlanta, GA: Department of Health and Human Services; April 2023.

3. Handsfield HH, Lipman TO, Harnisch JP, Tronca E, Holmes KK. Asymptomatic gonorrhea in men. N Engl J Med, 290(3), 117-123 (1974).

4. Peterman T, Tian L, Metcalf C et al. High incidence of new sexually transmitted infections in the year following a sexually transmitted infection: a case for rescreening. Ann Intern Med, 145(8), 564-572 (2006).

5. Harrison WO, Hooper MR, Wiesner PJ et al. A trial of minocycline given after exposure to prevent gonorrhea. N Engl J Med, 300(19), 1074-1078 (1979).

6. Wallin J. Gonorrhea in 1972: a 1-year study of patients attending the VD unit in Uppsala. Brit J Vener Dis, 51, 41-47 (1974).

7. Platt R, Rice PA, McCormack WM. Risk of acquiring gonorrhea and prevalence of abnormal adnexal findings among women recently exposed to gonorrhea. JAMA, 250(23), 3205-3209 (1983).

8. McCormack WM, Johnson K, Stumacher RJ, Donner A, Rychwalski R. Clinical spectrum of gonococcal infection in women. Lancet, 1(8023), 1182-1185 (1977).

9. Curran J, Rendtorff R, Chandler R, Wiser W, Robinson H. Female gonorrhea: its relation to abnormal uterine bleeding, urinary tract symptoms, and cervicitis. Obstet Gynecol, 45(2), 195-198 (1975).

10. Klein EJ, Fisher LS, Chow AW, Guze LB. Anorectal gonococcal infection. Ann Intern Med, 86, 340-346 (1977).

11. Wiesner PJ, Tronca E, Bonin P, Pedersen AHB, Holmes KK. Clinical spectrum of pharyngeal gonococcal infection. N Engl J Med, 288(4), 181-185 (1973).

12. Bro-Jorgensen A, Jensen T. Gonococcal pharyngeal infections: report of 110 cases. Brit J Vener Dis, 49, 491-499 (1973).

13. Svensson L, Westrom L, Ripa K, Mardh P. Differences in some clinical and laboratory parameters in acute salpingitis related to culture and serologic findings. Am J Obstet Gynecol, 138(7), 1017-1021 (1980).

14. Berger R, Alexander E, Harnisch J et al. Etiology, manifestations and therapy of acute epididymitis: prospective study of 50 cases. J Urol, 121(6), 750-754 (1979).

15. Holmes KK, Counts GW, Beaty HN. Disseminated gonococcal infection. Ann Intern Med, 74, 979-993 (1971).

16. Fleming D, Wasserheit J. From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to sexual transmission of HIV infection. Sex Transm DIs, 75(1), 3-17 (1999).

17. Thadepalli H, Rambhatla K, Maidman J, Arce JJ, Davidson EC Jr. Gonococcal sepsis secondary to fetal monitoring. Am J Obstet Gynecol, 126(4), 510-512 (1976).

18. U.S. Preventive Services Task Force. Screening for gonorrhea: recommendation statement. Ann Fam Med, 3, 263-267 (2005).

19. Van Der Pol B, Ferrero DV, Buck-Barrington L et al. Multicenter evaluation of the BDProbeTec ET system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens, female endocervical swabs, and male uerthral swabs. J Clin Microbiol, 39(3), 1008-1016 (2001).

20. Workowski, KA, Bachmann, LH, Chang, PA, et. al. Sexually Transmitted Infections Treatment Guidelines, 2021. MMWR Recomm Rep 2021; 70(No. 4): 1-187.

21. Centers for Disease Control and Prevention. Cephalosporin susceptibility among Neisseria gonorrhoeae isolates - United States, 2000-2010. MMWR, 60(26), 873-877 (2011).

22. Holmes KK, Levine R, Weaver M. Effectiveness of condoms in preventing sexually transmitted infections. Bull World Health Organ, 82(6), 454-461 (2004).

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Gonorrhea Fact Sheet from CDC. What is gonorrhea? How common is gonorrhea? How do people get gonorrhea? And more...

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1. Background

Gonorrhoea is a major public health concern, causing significant morbidity and socioeconomic consequences globally [ 1]. Neisseria gonorrhoeae has developed antimicrobial resistance (AMR) to all drugs available for treating gonorrhoea. In most countries, the only option for first-line empirical antimicrobial monotherapy is the extended-spectrum cephalosporin (ESC) ceftriaxone. Rare treatment failures of pharyngeal gonorrhoea with ceftriaxone and emergence of gonococcal strains exhibiting high-level clinical ESC resistance, combined with resistance to nearly all other antimicrobials, have resulted in development of global and regional action/ response plans [ 2- 6], and introduction of dual antimicrobial therapy (ceftriaxone 250 - 500 mg and azithromycin 1 - 2 g) in Europe, the USA and additional countries [ 3, 7, 8]. However, the susceptibility of gonococcal strains to ceftriaxone has decreased globally, resistance to azithromycin is already prevalent in many settings and concomitant resistance to both ceftriaxone and azithromycin has been identified in several settings [ 2, 3, 7, 9]. Consequently, these dual antimicrobial regimens might not be long-term solutions. Furthermore, these might not be affordable in many less-resourced settings, suffering from the highest gonorrhoea burden, and accordingly may not significantly mitigate AMR emergence and spread in a global perspective [ 3, 9]. Improved treatment and novel therapeutic antimicrobials are essential.

2. Conventional treatment of gonorrhoea

Public health control of gonorrhoea relies on effective antimicrobial treatment, that is, combined with prevention efforts, sensitive and specific diagnostics (index cases and traced sexual contacts) and epidemiological surveillance. Therapy is mostly given empirically, using first-line therapy according to management guidelines [ 7, 8] and before laboratory results are available. Ideally, the first-line therapy should be highly effective, widely available in appropriate quality, dose and cost, lack toxicity, possible to administer as single dose and cure > 95% of infected patients [ 4, 10]. Nevertheless, the evidence base for this > 5% threshold for changing recommended treatment is limited and levels of > 1 and > 3% AMR in high-frequency transmitting populations have also been suggested [ 10, 11]. Additional criteria, for example, prevalence, local epidemiology, transmission frequency, treatment strategies and cost, diagnostic tests and sexual contact tracing strategies, should ideally be considered when deciding when to alter recommended treatment. An identical AMR threshold and treatment strategy might not be the most cost-effective solution in all settings and populations [ 9, 12]. Unfortunately, the strategy of using a single antimicrobial in a single dose, slightly increased repeatedly over time due to resistance emergence, has likely selected for resistance [ 2, 3].

3. Future treatment of gonorrhoea

Future treatment must be in strict accordance with regularly updated management guidelines, which should be informed by quality assured surveillance of local AMR and treatment failures. Dual antimicrobial therapy (ceftriaxone and azithromycin [ 7, 8]), which also eradicates any concurrent chlamydial infection, should be considered in all settings where local quality assured AMR data do not support other therapeutic options. Nevertheless, in some settings compliance with therapy is a limited problem and, when evaluated, multiple doses of a single antimicrobial might also be considered in the future. Appropriate pharmacokinetic/pharmacodynamic simulations and clinical studies for both currently used and novel antimicrobials would be exceedingly valuable and, preferably, both monotherapy and dual therapy of urogenital and extragenital gonorrhoea with different combinations of antimicrobials and multiple doses, should be evaluated.

In the future, ideally treatment at first healthcare visit will be directed by rapid genetic point-of-care (POC) AMR tests, including simultaneous detection of gonococci. This POC test could directly provide a diagnosis and guide individually tailored treatments, which will ensure rational antimicrobial use (including sparing last-line antimicrobials), timely notification of sexual contacts and affect the public health control of both gonorrhoea and AMR. No commercially available gonococcal molecular diagnostic tests detect any AMR determinants. However, laboratory-developed molecular assays exist for detection of gonococcal AMR determinants [ 3, 13]. Unfortunately, for most genetic AMR determinants the sensitivity and specificity in their AMR prediction are relatively low (particularly for ESCs with their ongoing resistance evolution involving many different genes, mutations and their epistasis) [ 2, 3]. Genetic AMR testing will never entirely replace phenotypic AMR testing because the relationship between phenotype and genotype is not ideal and genetic methods can only detect previously known resistance determinants. However, enhanced research is imperative to continuously identify new resistance determinants and appropriately evaluate how current and future molecular AMR assays can supplement traditional AMR surveillance and guide individually tailored treatment [ 3, 13]. High-throughput genome sequencing, transcriptomics and other novel technologies might, initially in reference laboratories, revolutionize the genetic AMR prediction for both gonococcal isolates and primary samples positive for gonococci in genetic diagnostics.

4. Future treatment options for gonorrhoea

The dual antimicrobial treatment regimens with ceftriaxone and azithromycin [ 7, 8] are currently highly effective, however, as mentioned above these regimens might not be long-term solutions. Novel affordable treatments and/or antimicrobials for monotherapy or inclusion in dual treatment regimens, which might need to where feasible be considered for all newly developed antimicrobials, are essential.

A recent study evaluated two different novel dual antimicrobial regimens, that is, gentamicin (240 mg) plus azithromycin (2 g) and gemifloxacin (320 mg) plus azithromycin (2 g), for treatment of uncomplicated gonorrhoea. Microbiological cure was 100% with gentamicin + azithromycin and 99.5% with gemifloxacin + azithromycin, but adverse events were frequent with both regimens [ 14]. Generally, for dual antimicrobial therapy additional research is crucial to determine which antimicrobials (at what dosages) could be combined, and to appropriately document efficacy, side effects, pharmacokinetic/pharmacodynamic parameters, in vitro and in vivo synergistic relationship between antimicrobials, suppression of or increased resistance emergence (in gonococci and bystander organisms) and microbiological resistance breakpoints.

From a global public health perspective, where dual therapy is not affordable or feasible due to other reasons an effective antimicrobial monotherapy remain exceedingly valuable. 'Old' antimicrobials such as the aminocyclitol spectinomycin, aminoglycoside gentamicin, fosfomycin [ 15] and carbapenem ertapenem have been suggested and the in vitro susceptibility to all these appears relatively high. However, for empirical monotherapy all of these antimicrobials have shortcomings, for example, resistance is easily selected or resistance/decreased susceptibility already exist (spectinomycin, fosfomycin, ertapenem), mainly no recent appropriate clinical efficacy data for empiric monotherapy of urogenital and particularly extragenital gonorrhoea (gentamicin [mainly used in combination with doxycycline in Malawi], fosfomycin and ertapenem), evidenced suboptimal efficacy for treatment of pharyngeal gonorrhoea (spectinomycin) and lack of evidence-based correlates between MICs, pharmacokinetic/pharmacodynamic parameters and gonorrhoea treatment outcome (gentamicin, fosfomycin and ertapenem). Consequently, these antimicrobials are most likely mainly options for salvage therapy of ceftriaxone-resistant gonorrhoea and/or in novel dual antimicrobial treatment regimens [ 2, 3].

In recent years, several derivates of earlier developed antimicrobials have been evaluated in vitro against gonococcal strains [ 3]. The novel fluoroketolide solithromycin (family: macrolides) has high activity against gonococci, including ESC-resistant and multidrug-resistant (MDR) isolates. Nevertheless, gonococcal strains with high-level azithromycin resistance (MIC ≥ 256 µg/ml) appear to be solithromycin resistant (MICs = 4 - 32 µg/ml) [ 16]. An open-label, randomized, multicenter Phase III clinical trial is currently recruiting participants with uncomplicated gonorrhoea. The study aims to enroll 300 participants and treatment with solithromycin 1 g orally will be compared with a dual antimicrobial therapy regimen, that is, ceftriaxone 500 mg plus azithromycin 1 g ( www.clinicaltrials.gov). Also two 'bicyclic macrolides', that is, modithromycin (EDP-420) and EDP-322, recently showed potent in vitro activity against ESC-resistant and MDR gonococci. However, high-level azithromycin-resistant isolates (MIC ≥ 256 µg/ml) were resistant also to modithromycin and EDP-322 [ 17]. Several novel fluoroquinolones, that is, avarofloxacin (JNJ-Q2), sitafloxacin, WQ-3810 and delafloxacin, have a high in vitro activity against gonococci, including ciprofloxacin-resistant isolates. A Phase III clinical trial to compare delafloxacin (2 × 450 mg tablets administered once) with ceftriaxone (250 mg) for treatment of uncomplicated gonorrhoea in 757 patients was also designed, however, this study was recently terminated ( www.clinicaltrials.gov). The glycylcycline tigecycline and fluorocycline eravacycline (TP-434) (family: tetracyclines) appear also potent against gonococci in vitro. However, a limited proportion of administered tigecycline is excreted unchanged in urine, which questions the use in gonorrhoea treatment. Finally, the lipoglycopeptide dalbavancin and two new 2-acyl carbapenems SM-295291 and SM-369926 have also recently shown a high in vitro activity against a limited number of gonococcal isolates [ 3].

Clearly, novel antimicrobial targets, compounds and treatment strategies are essential to develop and evaluate. Therapeutic compounds with multiple targets might be important to suppress resistance emergence, however, further research is important to provide an appropriate evidence base for this. Interestingly, several antimicrobials or other compounds, using new targets or bactericidal or bacteriostatic strategies, have been designed in recent years. Several of these have also shown a high in vitro activity against gonococcal isolates. Some examples of these are new protein synthesis inhibitors such as pleuromutilin BC-3781 and the boron-containing inhibitor AN3365; LpxC inhibitors; species-specific FabI inhibitors such as MUT056399 and novel inhibitors of bacterial topoisomerases that target regions different from the fluoroquinolone-binding sites such as VT12-008911 and AZD0914 [ 3]. Regarding AZD0914, no resistance was indicated examining geographically and genetically diverse isolates including many fluoroquinolone, ESC and MDR isolates [ 18]. An open-label, randomized, multicenter Phase II clinical trial is also currently recruiting participants with uncomplicated gonorrhoea. The study aims to enroll 180 participants and treatment with AZD0914 2 g orally (n = 70) and AZD0914 3 g orally (n = 70) will be evaluated with ceftriaxone 500 mg (n = 40) as the active comparator ( www.clinicaltrials.gov). Interestingly, several examples of 'thinking out of the box' for future management of gonorrhoea have also been presented recently. These include efflux pump (particularly MtrCDE) inhibitors, particularly co-administered with antimicrobials, which increase the susceptibility to certain antimicrobials, innate host defense and toxic metabolites; non-cytotoxic nanomaterials; host defense peptides such as LL-37 (multifunctional cathelicidin peptide); molecules mimicking host defensins; factor H-Fc chimeric immunotherapeutic molecule [ 19] and IL-12 NanoCap, a biodegradable sustained-release formulation of human IL-12 that aims to be a therapeutic vaccine against gonococci ( http://therapyxinc.com/pipeline) [ 3].

5. Conclusions

Several of the recently developed novel antimicrobials or therapeutic compounds deserve increased attention for potential future treatment of gonorrhoea. Ideally, their activity should initially be evaluated in vitro against large collections of geographically, temporally and genetically diverse gonococcal isolates, including MDR strains, particularly with ESC resistance and azithromycin resistance. Understanding of the effects and biological fitness of current and future genetic resistance determinants ( in vitro induced/selected, including spontaneous resistance mutation frequency and mutant prevention concentration, and in vivo emerged) for these antimicrobials (both in gonococci and bystander organisms when treating gonorrhoea), time-kill curve analysis to evaluate bactericidal activity and clear correlates between genetic and phenotypic laboratory parameters and clinical treatment outcomes, would also be extremely valuable. Subsequently, in vivo evaluations ideally including mice model experiments and then appropriately designed, randomized controlled clinical trials evaluating parameters such as efficacy, safety, toxicity, cost, ideal dose and pharmacokinetic/pharmacodynamics data for genital and extragenital (especially pharyngeal) gonorrhoea. It is also important to determine the susceptibility of Chlamydia trachomatis and Mycoplasma genitalium to novel antimicrobials, which will indicate if the antimicrobials might be options for treatment of additional sexually transmitted infections (STIs) and STI syndromes, for example, urethritis and vaginal discharge. In general, for more sustainable gonorrhoea treatment in the future all newly developed antimicrobials might need to where feasible be considered for inclusion in a dual therapy regimen. Ideally, a gonococcal vaccine would be developed.

Footnotes
Declaration of interest
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

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Future treatment of gonorrhoea - novel emerging drugs are essential and in progress?

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Gonorrhoea is a major public health concern, causing significant morbidity and socioeconomic consequences globally [ 1]. Neisseria gonorrhoeae has developed antimicrobial resistance (AMR) to all drugs available for treating gonorrhoea. In most countries, the only option for first-line empirical antimicrobial monotherapy is the extended-spectrum cephalosporin (ESC) ceftriaxone.

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Suppression of Host Adaptive Immune Response

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Suppression of Host Adaptive Immune Response

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Abstract

Infection with Neisseria gonorrhoeae triggers an intense inflammatory response characterized by an influx of neutrophils in the genital tract, yet natural gonococcal infection does not induce a state of protective immunity. Our previous studies in a mouse model of N. gonorrhoeae infection demonstrated that TGF-β is involved in the suppression of adaptive immunity by this organism, but complete inhibition of TGF-β activity only partially reverses N. gonorrhoeae-mediated suppression of Th1 and Th2 responses. In this study, we show that N. gonorrhoeae strongly induced the production of IL-10 and type 1 regulatory T (Tr1) cells. Blockade of IL-10 and Tr1 cell activity enhanced both Th1/Th2-dependent adaptive immune responses and Th17-governed innate responses to N. gonorrhoeae. Treatment of mice with anti-IL-10 antibody during gonococcal challenge led to faster clearance of infection and induced protection against secondary infection, with the generation of circulating and vaginal anti-gonococcal antibodies. Our results suggest that inhibition of IL-10 and Tr1 cells affords a new approach to the treatment of gonorrhea and facilitates the development of specific protective immunity.

INTRODUCTION

Gonorrhea is the second most common sexually transmitted disease. The latest World Health Organization report estimates that more than 100 million new gonococcal infections occur worldwide every year, and the increasing resistance of Neisseria gonorrhoeae to even the latest generations of antibiotics is becoming problematic for effective treatment strategies. Ascending gonococcal infections occur frequently, particularly in women, leading to fallopian tube scarring, pelvic inflammatory disease, infertility, and risk of ectopic pregnancy, which can be life-threatening. Epidemiologic and clinical studies provide strong evidence that gonorrhea significantly increases the risk of acquisition and transmission of HIV.

N. gonorrhoeae typically triggers an intense inflammatory response characterized by an influx of neutrophils into the genital tract, yet natural gonococcal infection does not induce a state of specific protective immunity. Individuals with gonorrhea are usually not protected from reinfection, although one study reported partial protection against the same serovar of N. gonorrhoeae. This apparent lack of an adaptive immune response to N. gonorrhoeae probably contributes to the continuing prevalence of this sexually transmitted infection, and challenges the development of a vaccine against it. The conventional working hypothesis holds that N. gonorrhoeae can evade host immune defenses by multifactorial strategies including continuous changes in its surface antigenic structure, resistance to complement-mediated bacteriolysis, and possibly the production of IgA1 protease. However, increasing evidence indicates that N. gonorrhoeae as a highly adapted pathogen has evolved specialized mechanisms to proactively suppress specific immune responses and promote growth and persistence in the host. For example, it has been demonstrated that N. gonorrhoeae opacity (Opa) proteins are able to bind carcinoembryonic antigen-related cellular adhesion molecule (CEACAM)-1 on activated human CD4 T cells and down-regulate their activation and proliferation. Recently, Zhu et al reported that N. gonorrhoeae could inhibit both human and mouse antigen-dependent CD4 T cell proliferation through interactions with host antigen presenting dendritic cells.

Although it has been recognized that N. gonorrhoeae possesses the capacity to modulate host immune responses, the underlying mechanisms remain to be elucidated. Furthermore, comprehension of how this can be manipulated to generate protective adaptive immunity against the organism is limited. Our previous studies in a mouse model of gonococcal infection have demonstrated that N. gonorrhoeae elicits Th17 responses which are involved in the influx of neutrophils to the genital tract as well as the recruitment of other innate defense mechanisms. In contrast, N. gonorrhoeae can selectively suppress Th1 and Th2 activity of mouse CD4 T cells, and induction of TGF-β plays a critical role in these differential effects. Blockade of TGF-β diverts the pattern of host immune responses to N. gonorrhoeae and enhances specific protective immunity against the pathogen. However, we found that complete inhibition of TGF-β activity only partially reverses N. gonorrhoeae-mediated suppression of Th1 and Th2 cell proliferation and typical cytokine production. These results suggest that other unknown mechanisms also play an important role in the inhibitory effect of N. gonorrhoeae on Th1/Th2-mediated adaptive immune responses.

IL-10 is a regulatory cytokine produced by a variety of immune cells including activated T cells, monocytes/macrophages, B cells, dendritic cells, and mast cells, and it plays a major role in suppressing immune and inflammatory responses and maintaining specific T cell tolerance in both humans and mice. Type 1 regulatory T (Tr1) cells are one type of induced regulatory T cells, which inhibits Th1, Th2, and Th17 immunity through the production of immunosuppressive cytokines, mainly IL-10. Tr1 cells arise in the periphery when naïve CD4+ T cells are activated by tolerogenic antigen-presenting cells in the presence of IL-10. Therefore, the biological functions of IL-10 and Tr1 cells are closely related to each other. IL-10 is not only responsible for the regulatory effect of Tr1 cells but is also fundamental for their generation. Accumulating evidence indicates that IL-10 and Tr1 cells play a key role in regulating mucosal immune activation, for example, in the maintenance of gut immune homeostasis and tolerance to food antigens and enteric microbiota. In addition, IL-10 and Tr1 cells are exploited by many pathogens at mucosal sites to evade protective immunity, including Bordetella pertussis and Porphyromonas gingivalis. However, the potential role of IL-10 and Tr1 cells in genital tract infections has not been explored.

In this study, we show that, both in vitro and in vivo, N. gonorrhoeae strongly induced the production of IL-10 and Tr1 cells, which are critically involved in the suppression of adaptive immunity by the organism. Blockade of IL-10 and Tr1 cell activity significantly increased Th1, Th2, and Th17 responses to N. gonorrhoeae, and resulted in the generation of immune memory in a mouse model of vaginal gonococcal infection. Furthermore, our results revealed that inhibition of IL-10 in combination with inhibition of TGF-β had an additive effect in promoting protective immunity against N. gonorrhoeae.

RESULTS

N. gonorrhoeae elicits abundant production of IL-10 and Tr1 cells in vitro

To determine whether N. gonorrhoeae is capable of inducing IL-10 and Tr1 cells, we incubated mouse iliac lymph node (ILN) cells with N. gonorrhoeae (FA1090) in serum-free medium for various time periods. After 4 days, mouse lymphocytes stimulated with N. gonorrhoeae produced extremely high levels of IL-10, but not of Th1- or Th2-type cytokines, such as IL-12p70 or IL-4 (). Flow cytometric analysis showed that IL-10 was produced in multiple immune cell types, including CD4⁺, CD8⁺, CD19⁺, CD11b⁺, and CD11c⁺ cells (). IL-10 production increased with the time of incubation of N. gonorrhoeae (), and maximal stimulation was achieved at multiplicity of infection (MOI) 10:1; larger numbers of gonococci than MOI 100:1 tended to kill the cultures (data not shown). Prolonged stimulation for up to 14 d did not trigger Th1 or Th2 responses (). Similar levels of cytokine production were obtained when ILN cells were obtained from mice that had been infected in vivo, and then further cultured in vitro with N. gonorrhoeae; no significant increase in any cytokine occurred ( Supplementary Figure 1).

To assess whether high level IL-10 production is a common phenomenon following bacterial challenge, we also incubated mouse lymphocytes with another Gram-negative bacterium, Escherichia coli, under the same conditions. E. coli induced much lower levels of IL-10 ( p < 0.001) but more IL-12p70 ( p < 0.001) and IFN-γ ( p < 0.01), as well as IL-4 and IL-2 (). All these data suggest that infection with different bacteria results in different immune responses characterized by the production of Th lineage-associated cytokines, and show that N. gonorrhoeae potently elicits the immunosuppressive cytokine, IL-10.

Since N. gonorrhoeae induced high levels of IL-10 in the absence of IL-4 and IL-2, we investigated whether it stimulated the generation of Tr1 cells. Flow cytometry studies revealed that most IL-10-producing CD4⁺ T cells following gonococcal stimulation were IL-4 − and Foxp3 − (), which is consistent with a Tr1 cell classification. In contrast, N. gonorrhoeae did not significantly increase CD4⁺/Foxp3⁺ regulatory T cells (), and N. gonorrhoeae-induced Tr1 cells produced very low levels of TGF-β ().

To examine the direct effect of N. gonorrhoeae on CD4⁺ T cells, similar experiments were performed with purified CD4⁺ cells that were stimulated with N. gonorrhoeae in the presence of mitomycin C-inactivated antigen-presenting cells (APC). Similar results were observed. Supernatants from the cultures at day 4 showed abundant production of IL-10, but not IL-12p70, IFN-γ, IL-4 or IL-2 (). In contrast, CD4⁺ cells stimulated with N. gonorrhoeae in the absence of APC, or OT-II CD4⁺ cells stimulated with N. gonorrhoeae plus APC produced significantly less IL-10 ( Supplementary Figure 2). N. gonorrhoeae could also induce CD4⁺/IL-10⁺/IL-4 −/Foxp3 − Tr1 cells in purified CD4⁺ T cell cultures but to a lesser extent (), which suggests that IL-10 from other immune cell types contributes to the process.

To determine the production of cytokines in the mouse genital tract, vaginal explants were cultured with or without N. gonorrhoeae. Consistent with the above findings in lymphocyte cultures, IL-10 could be easily detected in the supernatants of vaginal explants stimulated with N. gonorrhoeae, whereas the production of other cytokines, including IL-12p70, IFN-γ, and IL-4, was minimal ().

N. gonorrhoeae induces IL-10 and Tr1 cells through multiple mechanisms

Our previous studies have demonstrated that gonococcal Opa proteins contribute to the induction of TGF-β and inhibition of Th1/Th2 responses by N. gonorrhoeae. In vivo, Opa proteins have been shown to promote persistence of N. gonorrhoeae in the lower genital tract of infected mice. We therefore investigated whether Opa proteins were involved in the elicitation of IL-10 and Tr1 cells in mouse lymphocytes by N. gonorrhoeae, using a mutant of N. gonorrhoeae (FA1090) in which all opa genes have been deleted. Previous colony dot-blot assays have shown that wild-type N. gonorrhoeae FA1090 expresses Opa A, B/D/G, and E/K, whereas the Opa-deficient mutant does not express any detectable Opa proteins. We cultured ILN cells or purified CD4⁺ T cells plus mitomycin C-inactivated APC with Opa-deficient or wild-type N. gonorrhoeae. In both whole lymphocyte and purified CD4⁺ T cell cultures Opa-deficient N. gonorrhoeae induced significantly less IL-10 ( p < 0.05) and Tr1 cells ( p < 0.05) in comparison with the wild-type strain ( Figure 2a and b ). However, the effect is modest, as in the absence of Opa expression N. gonorrhoeae still stimulated considerable levels of IL-10 and numbers of Tr1 cells, suggesting that other gonococcal factors and mechanisms also play an important role in this process.

Gonococcal lipooligosaccharide (LOS) is a critical virulence factor for N. gonorrhoeae and is a major inducer of the inflammatory cytokine response to the organism. We next determined whether generation of IL-10 and Tr1 cells was also dependent on LOS-TLR4 interactions. Iliac lymphocytes or purified CD4⁺ T cells plus inactivated APC from TLR4-normal C3H/FeJ or TLR4-deficient C3H/HeJ were stimulated with N. gonorrhoeae. Lack of TLR4 resulted in decreased IL-10 ( p < 0.001) and Tr1 cell ( p < 0.001) production in lymph node cell cultures ( Figure 2c and d ), but did not significantly affect N. gonorrhoeae-induced IL-10 and Tr1 cell production in purified CD4⁺ T cells ( Figure 2c and d ) possibly because the level of TLR4 expression on CD4⁺ cells is relatively low. Other cells in the ILN, such as B cells, may contribute significant amounts of IL-10 production.

IL-10 and Tr1 cells are involved in the suppression of Th1/Th2 immune responses by N. gonorrhoeae

We have previously demonstrated that N. gonorrhoeae selectively suppressed the development of Th1 and Th2 cells. Since N. gonorrhoeae can induce abundant production of IL-10 and Tr1 cells, we determined whether these factors are critically involved in the inhibitory effect of N. gonorrhoeae on Th1 and Th2 responses. Purified mouse CD4⁺ T cells were stimulated with anti-CD3 antibody under Th1-, Th2-, or Th17-polarizing conditions in the additional presence or absence of N. gonorrhoeae. IL-10 and Tr1 cell activities in the cultures were blocked by IL-10-neutralizing antibody at a dose determined to be effective without inducing toxicity. Flow cytometric analyses demonstrated that the addition of IL-10-neutralizing antibody significantly reversed N. gonorrhoeae-mediated suppression of Th1 and Th2 cell proliferation by 58.9±7.2% ( p < 0.01) and 37.6±5.3% ( p < 0.05), respectively (). Likewise, suppression of IFN-γ production in Th1 cells and of IL-5 production in Th2 cells were significantly reversed by IL-10-neutralizing antibody by 62.8±6.5% ( p < 0.001) and 43.5±5.7% ( p < 0.05), respectively (). Similar results were observed when Th1 or Th2 cells from IL-10-ko vs. wild-type mice were cultured with N. gonorrhoeae (data not shown). All these results indicate that IL-10 and Tr1 cells play an important role the inhibitory activity of N. gonorrhoeae against Th1/Th2-mediated adaptive immunity.

We next compared the effect of IL-10/Tr1 cells and TGF-β blockade on the suppression of Th1 and Th2 responses by N. gonorrhoeae. Both flow cytometry and ELISA showed that anti-IL-10 antibody reversed N. gonorrhoeae-mediated inhibition of Th1 cell development more effectively than anti-TGF-β antibody ( Figure 3a and b ). In contrast, anti-TGF-β antibody reversed the inhibition of Th2 cell development to a greater extent ( Figure 3a and b ). Inhibition of IL-10/Tr1 cells in combination with inhibition of TGF-β had an additive effect in reversing the suppression of Th1 and Th2 immunity by N. gonorrhoeae ( p < 0.05; ). However, in contrast to anti-TGF-β, anti-IL-10 antibody did not block Th17 cell development ( Figure 3a and b ).

Blockade of IL-10 and Tr1 cell activity enhances Th1/Th2 as well as Th17 immune responses to N. gonorrhoeae

IL-10 and Tr1 cells strongly inhibit Th1 and Th2 immunity. Unlike TGF-β, IL-10 is not involved in the generation of Th17 cells, and increasing evidence indicates that IL-10 and Tr1 cells also down-regulate Th17 immunity. We therefore hypothesized that blockade of IL-10 and Tr1 cell activity would enhance both Th1/Th2 and Th17 immune responses to N. gonorrhoeae. We then cultured ILN cells with gonococci in the presence of IL-10-neutralizing antibody or control IgG. Inhibition of IL-10 with a neutralizing antibody dramatically increased N. gonorrhoeae-induced IFN-γ production ( p < 0.001), and triggered the production of IL-12p70 and IL-4 ( p < 0.001; ). Anti-IL-10 antibody also increased IL-17 production ( p < 0.05) by lymphocytes stimulated with N. gonorrhoeae. The results were supported by RT-PCR studies of RORγt, T-bet, and GATA3 expression in N. gonorrhoeae-stimulated CD4⁺ T cells treated with anti-IL-10 antibody in comparison to control IgG (). Similar effects of IL-10 blockade were observed in vaginal explant cultures stimulated with N. gonorrhoeae, but the levels of IL-12p70, IFN-γ, IL-4 production that anti-IL-10 antibody could support in vaginal cultures were relatively low ( Supplementary Figure 3).

At present, it remains controversial whether IL-10 and TGF-β enhance the expression of each other. Our results revealed that neutralization of IL-10 and TGF-β in the cultures did not significantly decrease the production of each other (), which suggests that IL-10 and Tr1 cells, or TGF-β represent two distinct mechanisms involved in the suppressive effect of N. gonorrhoeae on Th1/Th2 immunity. Consistent with above results, both ELISA and RT-PCR analyses indicated that blockade of both IL-10 and TGF-β has an additive effect in promoting Th1 and Th2 immune responses against N. gonorrhoeae ( Figure 3c and d ).

IL-10 and Tr1 cell production in murine vaginal gonococcal infection

To determine whether N. gonorrhoeae induces IL-10 and Tr1 cells in vivo, we examined the local immune response in a mouse model of genital tract gonococcal infection. ILN and vaginas were removed from each of 5 mice per group at 1, 3, 5, 7, and 14 days after inoculation with N. gonorrhoeae or vehicle only. Single-cell suspensions were prepared from each specimen for evaluation by flow cytometry to detect the expression of IL-10, IL-4, and Foxp3. Starting on day 3 after inoculation, IL-10 production was observed in various immune cells present in the ILN, including CD4⁺, CD8⁺, CD19⁺, CD11b⁺, and CD11c⁺ cells (). IL-10 production peaked at day 5 and continued for the duration of infection. At day 5, 11.6% to 20.2% of the isolated immune cell types in the lymph nodes of infected mice were IL-10⁺, significantly higher than the levels in sham-infected controls (). Almost all IL-10⁺/CD4⁺ T cells were IL-4 − () and few were Foxp3⁺ (), a phenotype indicative of Tr1 cells. Similar results were observed in flow cytometric analysis of the infiltrating leukocytes in the vaginas (data not shown). The findings were further supported by quantitative RT-PCR analysis of mouse vaginal mRNA, which demonstrated that IL-10 was elevated by a factor of 10.4 ± 3.8 in N. gonorrhoeae-infected mice relative to control mice ( p < 0.001). All these data indicated that N. gonorrhoeae elicits abundant production of IL-10 and Tr1 cells in vivo in a murine model of vaginal gonococcal infection.

ELISA analysis of vaginal wash, saliva, and serum of N. gonorrhoeae-infected and sham-infected mice showed a peak level of IL-10 (~60-90 pg/ml) in vaginal washes from infected mice at days 3 to 5 after inoculation. However, IL-10 was not detected in saliva or serum samples of any mouse at any time point.

Blockade of IL-10 and Tr1 cell activity enhances both Th1/Th2-mediated adaptive immune responses and Th17-governed innate responses in vaginal gonococcal infection

Given the strong induction of IL-10 and Tr1 cells by N. gonorrhoeae and their known suppressive effect on Th1/Th2 immunity, we hypothesized that blockade of IL-10 and Tr1 cells would elicit host Th1 and Th2 adaptive responses to gonococcal infection and enhance bacterial clearance. To test this, groups of female BALB/c mice were vaginally infected with N. gonorrhoeae, and the bacterial burden was monitored daily by vaginal swab culture. The mice were injected with 300 μg of IL-10 neutralizing antibody or control IgG on days −1 and 0 and every two days thereafter. Preliminary experiments showed this dose to be optimal without generating side-effects. The duration of gonococcal infection in control IgG-treated mice was ~15 days (). Anti-IL-10 treatment significantly reduced the recoverable gonococcal load starting from day 4, and led to faster clearance of the infection ( F 1,238 = 69.20; p < 0.001; ). Flow cytometric analysis demonstrated that inhibition of IL-10 and Tr1 cells resulted in enhanced Th1 and Th2 immune responses, indicated by significantly increased numbers of IFN-γ⁺/CD4⁺ ( p < 0.01) and IL-4⁺/CD4⁺ T cells ( p < 0.05) in the ILN from treated mice in comparison to those from control mice (). In contrast to the effect of anti-TGF-β observed in our previous study, anti-IL-10 did not hinder the Th17 response; instead it elevated the number of IL-17⁺/CD4⁺ T cells ( p < 0.05) in ILN (). Similar results were observed in vaginas of the same mice. RT-PCR analyses showed that IFN-γ ( p < 0.001), IL-4 ( p < 0.01), and IL-17 ( p < 0.05) mRNA expressions were all enhanced in the infected mice following anti-IL-10 antibody treatment (). In addition, anti-IL-10 increased Gr-1⁺ neutrophil ( p < 0.01), CD8⁺ ( p < 0.05), and CD19⁺ cell ( p < 0.01) influx into the genital tract (). Consistent with the in vitro experiments, these in vivo findings revealed that inhibition of IL-10/Tr1 cell and TGF-β activities had an additive effect in overcoming the suppression of Th1 and Th2 immunity by gonococci ( Figure 4e and f ). Treatment of infected mice with both anti-IL-10 and anti-TGF-β further accelerated clearance of N. gonorrhoeae ( F 1,238 = 13.31; p < 0.01; ).

To confirm the importance of IL-10 and Tr1 cells in the response to vaginal infection with N. gonorrhoeae, we repeated the experiment using IL-10-knockout (IL-10 KO; C57BL/6 background) mice, which lack the capability to generate IL-10 and Tr1 cells. Gonococcal infection in wild-type C57BL/6 mice lasted about 2 weeks, whereas IL-10 KO mice started to show diminished gonococcal recovery from day 3 and had cleared the infection by day 7 ( F 1,224 = 61.74; p < 0.001; ).

Since IL-10 was produced by a variety of immune cell types in addition to Tr1 cells, we then investigated the specific role of Tr1 cells in an adoptive transfer experiment. IL-10- or N. gonorrhoeae-induced Tr1 cells were generated by prolonged culture of CD4⁺ cells from BALB/c mice in the presence of IL-10 or N. gonorrhoeae, as described in Methods. Groups of BALB/c mice infected with N. gonorrhoeae were injected intravenously on days 0 and 3 with 1 × 10 ⁶ Tr1 or control CD4⁺ T cells. Transfer of N. gonorrhoeae-induced ( F 1,252 = 64.03; p < 0.01) but not IL-10-induced ( F 1,252 = 1.06; p = 0.31) Tr1 cells elevated the recoverable gonococcal burdens in the treated mice from day 5 through 13 after inoculation (). However, the transfer of exogenous Tr1 cells did not prolong the duration of infection, possibly because large numbers of these cells were induced endogenously in the animals by infection with N. gonorrhoeae.

Treatment with anti-IL-10 antibody protects mice against secondary N. gonorrhoeae infection

Since inhibition of IL-10 and Tr1 cells elicited the development of Th1/Th2 adaptive responses to primary gonococcal infection, we then assessed whether the treatment resulted in the generation of immune memory and protective immunity. Reinfection studies were performed in which groups of mice treated with anti-IL-10 or control IgG during primary infection with N. gonorrhoeae were allowed to recover and then treated with ceftriaxone on day 15 to ensure complete clearance of the infection. An additional group of sham-infected mice treated with anti-IL-10 was used to evaluate the possible persistent effect of anti-IL-10 antibody in the absence of infection. Five to six weeks later, all mice were inoculated with N. gonorrhoeae of the same strain without any further treatment. Consistent with previous studies, the duration and bacterial burden of secondary infection in previously control IgG-treated mice were the same as for the primary infection of age-matched naïve mice ( F 1,294 = 3.25; p = 0.09; ). In contrast, anti-IL-10 treatment during primary N. gonorrhoeae infection protected the mice against secondary infection. Reinfected mice that had been treated with anti-IL-10 antibody during the primary infection recovered significantly faster than controls ( F 1,294 = 99.13; p < 0.001; ). Interestingly, previous anti-IL-10 treatment of sham-infected mice did not induce protection against subsequent infection ( F 1,294 = 0.44; p = 0.51; ). We also found that treatment with anti-IL-10 combined with anti-TGF-β during primary gonococcal infection protected the mice more effectively than treatment with anti-IL-10 alone ( F 1,294 = 10.68; p < 0.05; ).

Flow cytometric and RT-PCR studies of ILN and vaginas taken on day 5 indicated that the protective effect of previous anti-IL-10 treatment on secondary gonococcal infection was also associated with enhanced Th1 (IFN-γ) ( p < 0.001) and Th2 (IL-4) ( p < 0.01) responses ( Figure 5b and c ). However, Th17 response (IL-17) in the reinfected mice with previous anti-IL-10 treatment was similar to those receiving control IgG treatment.

We then tested whether inhibition of IL-10 and Tr1 cells during N. gonorrhoeae infection led to the production of anti-gonococcal antibodies. IgM antibodies showed low levels and little difference between experimental groups (data not shown). No salivary gonococcus-specific antibody was detected in any group of mice (data not shown). Without anti-IL-10 treatment, neither primary nor secondary N. gonorrhoeae infection of mice induced the production of significant gonococcus-specific IgA or IgG antibody responses in either serum or vaginal wash ( Figure 6a-d ). There was also no significant increase in specific IgA or IgG antibody responses in serum or vaginal wash to primary infection with anti-IL-10 treatment ( Figure 6a and b ). However, there was a specific antibody response in anti-IL-10-treated mice after they were reexposed to N. gonorrhoeae. Gonococcus-specific IgG antibodies in vaginal wash and serum of reinfected mice with previous anti-IL-10 treatment were significantly higher than those of controls ( p < 0.01; Figure 6c and d ). However, anti-IL-10 did not increase the production of specific IgA antibody in either serum or vaginal wash. Furthermore, we found that inhibition of both IL-10 and TGF-β during primary N. gonorrhoeae infection had an additive effect on enhancing the gonococcus-specific IgG antibody responses in both vaginal wash and serum to subsequent infection ( p < 0.05; Figure 6c and d ).

DISCUSSION

We have previously shown that vaginal infection of mice with N. gonorrhoeae selectively elicits IL-17-dependent innate responses and concomitantly suppresses Th1/Th2-driven adaptive immune responses through mechanisms that depend on TGF-β. Administration of anti-TGF-β antibody alleviates this suppression and permits the development of Th1 and Th2 responses, as well as the establishment of immune memory and the generation of anti-gonococcal antibodies, thereby accelerating clearance of a primary gonococcal infection and protecting against a subsequent infection.

The involvement of TGF-β in these effects suggests a role for regulatory T cells, which have been previously reported in the mouse model of genital gonococcal infection. However, we noted that N. gonorrhoeae did not strongly induce expression of the nuclear transcription factor, Foxp3, that is associated with conventional induced T-regulatory (Treg) cell function. We now show that N. gonorrhoeae induced very high levels of IL-10 and Tr1 cells both in cultured lymphoid cells in vitro, and in cells recovered from mice vaginally infected with N. gonorrhoeae. Since OT-II CD4⁺ cells stimulated with N. gonorrhoeae, or wild-type CD4⁺ cells stimulated with N. gonorrhoeae in the absence of APC produced significantly less IL-10, it is likely that a significant proportion of N. gonorrhoeae-activated IL-10-producing CD4⁺ cells are gonococcus-specific. The generation of IL-10 appeared to be a specific effect of N. gonorrhoeae, as culture of cells with another gram-negative bacterium, E. coli, induced much lower levels of IL-10 and much more IFN-γ and IL-12. An Opa-deficient mutant of N. gonorrhoeae induced slightly lower levels of IL-10 in mouse cells in vitro, suggesting that other gonococcal components probably play an important role in this process. However, we could not determine whether a similar outcome occurs in vivo because this Opa-deficient mutant appears to have growth defects which compromise its ability to infect mice. Moreover, it is unclear at present what receptors are engaged by gonococcal Opa proteins, as murine CEACAM receptors (unlike human CEACAMs) do not bind Opa. Unfractionated lymph node cells from TLR4-deficient mice were significantly less able to generate IL-10 in response to stimulation with N. gonorrhoeae, implying that gonococcal LOS contributes to this effect, but purified CD4⁺ T cells (plus APC) did not replicate this effect, suggesting that LOS-responsive cells were not sufficiently represented in these cultures. Whereas TLR4 is expressed on myeloid cells including APC, it is not present at high levels on murine T cells. We found previously that murine B cells which are known to express TLR4 contribute to the LOS-stimulated generation of Th17 responses. Others have found that TLR4-deficient mice infected with N. gonorrhoeae show higher peak bacterial loads and diminished Th17-related cytokine responses, but IL-10 was not examined. However, as TLR4-deficiency impacts on many other aspects of the inflammatory response to infection in addition to IL-10 production, this outcome is not necessarily at variance with our findings.

Although many cell types secreted IL-10 in response to gonococcal stimulation, we found that the IL-10-producing CD4⁺ T cells were mostly IL-4 −/Foxp3 −, a phenotype characteristic of Tr1 cells . This subset of regulatory T cells is known to suppress Th1, Th2, and also Th17 cells. Anti-IL-10 antibody reversed the suppression of Th1 and Th2 cells by N. gonorrhoeae without affecting the induction of Th17 cells. Anti-TGF-β antibody had an additive effect to anti-IL-10 on the reversal of Th1 and Th2 suppression, but it also counteracted the induction of Th17 cells which require TGF-β for their generation.

These findings from in vitro experiments were reflected in the tissues of mice infected with N. gonorrhoeae in vivo. Thus lymphoid cells isolated from vaginal tissue and from the ILN, which drain the genital tract, of N. gonorrhoeae-infected mice showed production of IL-10 by CD4⁺ and CD8⁺ T cells, B cells, as well as both CD11b⁺ and CD11c⁺ cells which are taken to be macrophages and dendritic cells (DC) respectively. The selective induction of IL-10 in DC by N. gonorrhoeae has recently been reported. Furthermore, the CD4⁺ cells that produced IL-10 in vivo were largely IL-4 − and Foxp3 −, corresponding to the Tr1 phenotype. Accordingly, treatment of mice with blocking antibody to IL-10 during vaginal gonococcal infection accelerated the clearance of the infection, and enhanced the generation of Th1 and Th2 cells without impairing the generation of Th17 cells. Co-administration of anti-TGF-β antibody with anti-IL-10 resulted in even faster clearance. In addition, IL-10-deficient mice were significantly more resistant to vaginal gonococcal infection than their wild-type counterparts. This phenotype is similar to that of IL-22-knockout mice, which are also resistant to vaginal gonococcal infection . Beyond noting that IL-22 is structurally related to IL-10, shares the IL-10R2 signaling component in its receptor, and signals through Jak-STAT pathways, we have no satisfactory explanation for the latter observation, as the two cytokines target different cells and have quite different biological effects.

To test directly the involvement of Tr1 cells in the suppression of immune responses by N. gonorrhoeae, we performed an adoptive transfer experiment. ILN cells from N. gonorrhoeae-infected mice were cultured for a prolonged period with gonococci of the same strain in the presence of anti-CD3 antibodies, which led to the generation of gonococcus-specific Tr1 cells. Adoptive transfer of these cells into congenic mice that were infected with N. gonorrhoeae impaired early reduction of the bacterial load. Non-specific Tr1 cells generated by culture with IL-10 did not have this effect. However, transfer of gonococcus-specific Tr1 cells did not prolong the total course of infection, and we ascribe that to the endogenous generation of Tr1 cells as a consequence of infection. In these circumstances, it seems likely that the adoptive transfer of additional, exogenously generated Tr1 cells served to suppress the early response but did not alter the ultimate outcome.

We previously demonstrated that treatment of mice with blocking antibodies to TGF-β during primary gonococcal infection leads to the establishment of Th1- and Th2-driven immune memory that can be recalled upon subsequent re-infection with N. gonorrhoeae . As a result, the second infection is cleared more effectively, and anti-gonococcal IgA and IgG antibodies are found in the serum and genital secretions-neither of which outcomes occurs normally in reinfected mice. We found similar development of protective anamnestic immune responses, including anti-gonococcal IgG antibodies, when mice that were treated with anti-IL-10 antibody during primary infection were challenged a second time but without further treatment with anti-IL-10 antibody. Moreover, treatment with anti-TGF-β in addition to anti-IL-10 antibody increased the effect. Similar to blockade of TGF-β, which promotes both Th1 and Th2 anamnestic responses, blockade of IL-10 enhanced Th1 and Th2 responses measured by the expression and production of IFN-γ and IL-4 in the genital tract tissues and draining ILN. However, in contrast to anti-TGF-β which facilitates the generation of both IgG and IgA anti-gonococcal antibody responses, anti-IL-10 treatment enhanced only IgG antibody responses in serum and vaginal secretion, and unlike anti-TGF-β it did not inhibit the IL-17 response.

From these findings and our previous studies, we conclude that N. gonorrhoeae suppresses Th1- and Th2-driven adaptive immune responses against itself by mechanisms that depend upon TGF-β and IL-10, and that involve the generation of Tr1 cells which are known to inhibit Th1 and Th2 cells. This induced, specific immunosuppression can be reversed by blockade of IL-10 and/or TGF-β, thereby allowing the induction of Th1 and Th2 cells, the establishment of immune memory, the generation of specific anti-gonococcal antibodies, and the development of protective immunity. This has not previously been accomplished in models of genital tract infection with N. gonorrhoeae.

We have proposed that the suppression of Th1- and Th2-driven specific immune responses coupled with the selective elicitation of Th17-dependent innate immune responses is an adaptive strategy that enables N. gonorrhoeae to colonize its host by proactively directing the pattern of host immune responses in its own favor. N. gonorrhoeae appears well able to resist innate defense mechanisms driven by Th17 responses, as it can survive within neutrophils and resist antibacterial proteins such as defensins by means of a multi-drug exporter mechanism. Under this hypothesis we propose that Th1/Th2-driven adaptive immune responses, such as the generation of specific anti-gonococcal antibodies, provide the key additional factor that arms host defenses effectively against N. gonorrhoeae. It is unclear at present whether Th17-driven responses, such as the neutrophil influx, are essential for clearance of vaginal gonococcal infection in mice. IL-17 receptor A-knockout mice, or normal mice treated with IL-17-blocking antibody, show prolonged infection and diminished neutrophil influx into the genital tract . However, the association of these two effects may be coincidental, because the neutrophil influx varies between different strains of mice regardless of the clearance kinetics. Moreover, in our studies of murine vaginal gonococcal infection, clearance still occurred in IL-17 receptor-knockout mice despite the absence of a neutrophil influx . It is likely that numerous factors are responsible for the elimination of vaginal gonococcal infection in mice, including not only host responses to the infection, but also the fact that N. gonorrhoeae is an exclusively human pathogen that has become evolutionarily well-adapted to its sole natural host and is not ordinarily capable of infecting mice. However, it is possible that these effects are limited to the artificial murine infection, and therefore it is critical to determine the extent to which they apply to the natural human infection. It has already been reported that humans infected with gonorrhea have elevated serum IL-17 and IL-23, but it is not clear whether this is a consequence of infection or a predisposing factor in acquiring the infection.

The preponderance of clinical experience shows that episodes of gonorrhea do not induce effective immunity against re-infection, as gonorrhea can be re-acquired with no apparent reduction in severity or duration of disease. The conventional explanations adduced for this situation include the extraordinary capacity of N. gonorrhoeae for antigenic variation involving most of its surface antigens, coupled with numerous mechanisms for inhibiting complement activation and resisting cytolysis. It is also becoming clear that N. gonorrhoeae can survive phagocytosis by neutrophils, the predominant cells found in the exudates that are characteristic of symptomatic gonorrhea, by interfering with both oxygen-dependent and oxygen-independent intracellular killing. While all of these mechanisms undoubtedly contribute to the success of N. gonorrhoeae as a pathogen, we propose that it also suppresses specific adaptive immune responses, including antibody development, that might be capable of eliminating it.

Our findings reported here and previously support this concept and indicate some of the mechanisms whereby this is achieved. Our findings are complemented by a recent report that N. gonorrhoeae interacts with DC to induce the production of not only IL-10 but also programmed death ligand 1 (PDL1). As a result, such gonococcus-exposed DCs suppress antigen-induced proliferation of CD4⁺ T cells. It had previously been shown that N. gonorrhoeae can inhibit the proliferation of human CD4⁺ T cells through the binding of gonococcal Opa proteins to it receptor, CEACAM-1, which is expressed on these cells. A similar effect on human B cells was also reported . Thus it appears that N. gonorrhoeae can interact with host immune cells at multiple levels, i.e., with antigen-presenting cells, T-helper cells, and B cells, as well as Tr1 cells, to suppress the development of specific immune responses. We propose that if specific immune responses to N. gonorrhoeae were allowed to proceed in the normal manner, then the effector products of such immune activation, particularly including anti-gonococcal antibodies, might be able to eliminate the infection. Moreover, the concomitant establishment of immune memory might provide protection against re-infection, assuming a sufficient degree of antigenic similarity between the original and new infecting strains. Possibly this occurs eventually in natural human infections if they are allowed to proceed untreated, and anecdotal evidence from the pre-antibiotic era suggests that gonorrhea is ultimately self-limiting. However, it is completely unethical to test such speculations directly in humans. Instead, our evidence from experimental murine infections indicates that it is possible to immunologically reverse the natural capacity of N. gonorrhoeae to suppress Th1- and Th2-mediated specific immune responses against it, and allow the development of protective immune responses that not only accelerate clearance of an existing infection (therapy) but also generate protection against future infection (prophylaxis). In our experiments so far these include the systemic administration of blocking antibodies against TGF-β and IL-10. Ongoing studies are aimed at determining whether these approaches can be developed into more practical interventions against genital tract infection with N. gonorrhoeae.

METHODS

Mice

All mice, including wild-type BALB/c and C57BL/6 mice, C3H/HeJ (TLR4-deficient), C3H/FeJ (TLR4-normal), B6.129P2-Il10tm1Cgn/J (IL-10 KO), and B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II) mice on a C57BL/6 background, were purchased from Jackson Laboratories (Bar Harbor, ME). BALB/c mice were used for the experiments unless otherwise specified. Mice were maintained under standard conditions in the Laboratory Animal Facility at the University at Buffalo, which is fully accredited by AAALAC. All animal use protocols were approved by the Institutional Animal Care and Use Committee of the University at Buffalo.

Bacteria

N. gonorrhoeae FA1090 and an Opa-deletion mutant of FA1090 were kindly provided by Dr Janne Cannon (University of North Carolina at Chapel Hill). Bacteria were cultured on GC agar supplemented with hemoglobin and Isovitalex (BD Diagnostic Systems, Franklin Lakes, NJ) and the resultant growth was checked for colony morphology consistent with Opa protein and pilus expression. Bacteria were harvested from plates and the cell density was determined as detailed previously.

Mouse vaginal infection model

Female mice between 7 and 9 weeks old were infected with live N. gonorrhoeae FA1090 as previously described, with the modification that water-soluble estradiol was administered on days −2, 0, and 2. Vaginal mucus was quantitatively cultured daily on GC agar supplemented with selective antibiotics to determine the bacterial colonization loads. In some experiments, mice were injected subcutaneously on days −1, 0, and every two days thereafter, with 300 μg IL-10 or/and TGF-β neutralizing mAb or control mouse IgG purchased from Bio X Cell (West Lebanon, NH).

Lymphocyte isolation and culture

Mononuclear cells were isolated from aseptically harvested ILN using Histopaque 1083 (Sigma-Aldrich, St Louis, MO) density gradient centrifugation. CD4⁺ T cells were purified by magnetic sorting (MACS) through negative selection using a Dynal CD4 cell isolation kit (Invitrogen, Carlsbad, CA). Cells were cultured in 24-well culture plates at a density of 1 × 10 ⁶ cells/ml with either no stimulus or N. gonorrhoeae cells at indicated MOI. For some purified CD4⁺ T cell cultures, equal numbers of APC (mitomycin C-inactivated splenocytes) were added. X-VIVO serum-free medium (Lonza, Walkersville, MD) with penicillin/streptomycin was used for all experiments. For some experiments, 40 μg/ml mouse IL-10 and/or TGF-β neutralizing mAb (Bio X Cell) was added.

Th cell differentiation

Naïve purified CD4⁺ T cells (1 × 10 ⁶ cells/ml) were cultured in 24-well tissue culture plates, and stimulated with 1.0 μg/ml plate-bound anti-CD3 antibody (eBiosciences, San Diego, CA) in complete X-VIVO medium. Differentiation of Th1, Th2 and Th17 cells was induced by the addition of IL-12 and anti-IL-4 antibody, IL-4 and anti-IFN-γ antibody, or TGF-β and IL-23 plus anti-IFN-γ and anti-IL-4 antibodies, respectively, as detailed previously. In each condition, the cells were cultured in the absence or presence of N. gonorrhoeae FA1090 (MOI 50:1) and mitomycin C-inactivated APCs for 4 d. Supernatants and cells were then collected for ELISA and flow cytometry analyses. In some experiments, 40 μg/ml anti-IL-10 and/or anti-TGF-β mAb was added as indicated.

Vaginal explant culture and cell isolation

Genital tracts were dissected out aseptically and washed with Hanks's buffered salt solution. The tissue was manually separated into about 1 mm pieces, washed again and weighed. Equal weights were added to each well of 24-well cell culture plates. Tissue was incubated in X-VIVO serum-free medium supplemented with penicillin, streptomycin, and fungizone. Vaginal single-cell suspensions were prepared by protease digestion of excised vaginal tissue, as described previously.

In vitro Tr1 cell induction

For generation of IL-10-induced Tr1 cells, CD4⁺/CD62L⁺ T cells were isolated from naïve BALB/c mouse ILN by FACS (FACSAria; Becton-Dickinson). The sorted cells were cultured and stimulated repeatedly for 3 weeks with anti-CD3 antibody (eBiosciences) in the presence or absence (control) of IL-10 (10 ng/ml). Medium with antibodies, cytokines, and stimulants was changed twice a week, and IL-2 (50 units/ml) was added to the cultures after 2 weeks. For generation of N. gonorrhoeae-induced Tr1 cells, ILN cells obtained from N. gonorrhoeae-infected mice were similarly stimulated for 3 weeks with anti-CD3 in the presence of N. gonorrhoeae. At the end of stimulation, CD4⁺ cells were purified by MACS for adoptive transfer.

Cytokine ELISA

IL-10, TGF-β, IFN-γ, IL-12p70, IL-4, IL-5, and IL-17A levels were measured in triplicate using ELISA kits purchased from eBioscience.

Flow cytometry

Cells were washed with staining buffer twice, then incubated with the indicated antibodies for 30 min on ice, washed twice, and analyzed on a FACSCalibur cytometer. For intracellular staining, cells were first fixed with Cytofix/Cytoperm (eBioscience). Antibodies to mouse CD4, CD8, CD19, CD11b, CD11c, NKG2D, Gr-1, IFN-γ, IL-4, IL-17A, and Foxp3 conjugated with FITC, PE, or allophycocyanin were purchased from eBioscience. PE-conjugated anti-TGF-β antibody was obtained from IQ Products (Groningen, The Netherlands).

Proliferation assays

Cells were labeled with carboxymethyl fluorescein succinimide ester (CFSE; Sigma-Aldrich) according to a previous protocol. CFSE-labeled cells were then washed twice in PBS, recounted, and stimulated as described above. Cultured cells were harvested and then stained with allophycocyanin-conjugated anti-mouse CD4 antibody. The data were acquired by gating on the CD4⁺ cell populations in a FACSCalibur cytometer. The sequential loss of CFSE fluorescence was used to measure cell proliferation.

Real-time RT-PCR

Total cellular RNA of MACS-purified cultured CD4⁺ T cells or whole vaginas harvested from the mice was isolated with RNeasy Mini Kits (Qiagen, Valencia, CA), and was transcribed to cDNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time RT-PCR was performed on an iCycler iQ detection system (Bio-Rad) using Sybergreen (Bio-Rad) for real-time monitoring of the PCR. The primers used were as follows: T-bet, 5′-GGA GCG GAC CAA CAG CAT CG-3′; 5′-GCG GGC GGC GGG AAG AAC T-3′; GATA3, 5′-CCG AAA CCG GAA GAT GTC TA-3′; 5′-AGG GCT CTG CCT CTC TAA CC-3′; ROR-γt, 5′-CCG CTG AGA GGG CTT CAC-3′, 5′-TGC AGG AGT AGG CCA CAT TAC-3′; IFN-γ, 5′-TAC TGC CAC GGC ACA GTC ATT GAA-3′, 5′-GCA GCG ACT CCT TTT CCG CTT CCT-3′; IL-4, 5′-GAA GCC CTA CAG ACG AGC TCA-3′; 5′-ACA GGA GAA GGG ACG CCAT-3′; IL-17A, 5′-TCA GGG TCG AGA AGA TGC TG-3′; 5′-TTT TCA TTG TGG AGG GCA GA-3′; IL-10, 5′-GCT CCT AGA GCT GCG GAC T-3′; 5′ TGT TGT CCA GCT GGT CCT TT-3′; β-actin, 5′-CCT AAG GCC AAC CGT GAA AAG-3′, 5′-GAG GCA TAC AGG GAC AGC ACA-3′. Relative quantification of target genes was analyzed based on the threshold cycle (Ct) determined by Bio-Rad iQ5 optical system software.

Assay of serum and mucosal antibodies

Samples of saliva, vaginal wash, and serum were collected from individual mice on day 15 post-inoculation. Gonococcus-specific IgA, IgG, and IgM in saliva, sera, and vaginal washes were measured by ELISA on plates coated with whole gonococci (FA1090). Total IgA, IgG, and IgM concentrations in secretions were assayed by ELISA on plates coated with anti-IgA, -IgG, or -IgM antibodies (Southern Biotech, Birmingham, AL). H5 mouse monoclonal antibody (specific for FA1090 porin; kindly provided by Ann E. Jerse, Uniformed Services University of the Health Sciences) or affinity-purified mouse IgA, IgG, and IgM (Southern Biotech) were used to establish standard curves. Bound antibodies were detected by alkaline phosphatase-conjugated goat anti-mouse IgA, IgG, or IgM antibody (Southern Biotech) and p-nitrophenylphosphate substrate (Southern Biotech). Plates were read in a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA), and calibration curves for interpolation of unknowns were constructed using SoftMax software (Molecular Devices).

Statistical analysis

Data are expressed as the means ± standard errors of the means (SEM). Data on the effect of anti-IL-10 or/and anti-TGF-β versus control IgG treatments on mouse vaginal N. gonorrhoeae infection were analyzed using two-way analysis of variance (ANOVA) with Fisher's protected least significant difference post hoc tests for multiple comparisons. Unpaired two-tailed t tests were used to compare the mean values between two groups. P < 0.05 was considered statistically significant.

Acknowledgments

We are grateful to the assistance of the Confocal Microscope and Flow Cytometry Facility in the School of Medicine and Biomedical Sciences, University at Buffalo. We thank Dr. Ann E. Jerse for helpful advice and valuable discussions during the course of this work. This work was supported by NIH grant AI074791 from the National Institute of Allergy and Infectious Diseases, and by a grant from The John R. Oishei Foundation, Buffalo, NY.

Footnotes
DISCLOSURE
The authors declared no conflict of interest.

References

1. World Health Organization. Global action plan to control the spread and impact of antimicrobial resistance in Neisseria gonorrhoeae. WHO; Geneva, Switzerland: 2012.

3. Hook EW, Handsfield HH. Gonococcal infections in the adult. In: Holmes KK, Sparling PF, Mårdh P-A, Lemon SM, Stamm WE, Piot P, Wasserheit JN, editors. Sexually Transmitted Diseases. 3. McGraw-Hill Health Professions Division; New York: 1999. pp. 451-466.

5. Russell MW, Hook EW., 3rd . Gonorrhea. In: Barrett ADT, Stanberry LR, editors. Vaccines for biodefense and emerging and neglected diseases. Academic Press; London: 2009. pp. 963-981.

10. Lewis LA, Burrowes E, Rice PA, Ram S. Interactions of Neisseria with complement. In: Genco CA, Wetzler L, editors. Neisseria: Molecular Mechanisms of Pathogenesis. Caister Academic Press; Norfolk, UK: 2010. pp. 123-144.

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Suppression of host adaptive immune responses by Neisseria gonorrhoeae: role of interleukin 10 and type 1 regulatory T cells

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Infection with Neisseria gonorrhoeae triggers an intense inflammatory response characterized by an influx of neutrophils in the genital tract, yet natural gonococcal infection does not induce a state of protective immunity. Our previous studies in a mouse model of N.

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Diversion of Immune Response to Neisseria Gonorrhoeae

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Diversion of Immune Response to Neisseria Gonorrhoeae

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Diversion of the immune response to Neisseria gonorrhoeae from Th17 to Th1/Th2 by treatment with anti-transforming growth factor β antibody generat... - PubMed - NCBI

# data[].attributes.attachment_link.description
Immunity to N. gonorrhoeae is a topic that until recently has been hard to define, because there is currently no clearly established state of immunity to gonorrhea in humans, which are the only natural host for this infection. Furthermore, until recently, only one animal model of genital tract infection has been developed, known as the estradiol-treated female mouse model.

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Pathogenesis of : neutrophilic host response, sustained infection, and clinical sequelae Neisseria gonorrhoeae : neutrophilic host response, sustained infection, and clinical sequelae in the female reproductive tract: neutrophilic host response, sustained infection, and clinical sequelae

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Gonorrhea is a major global health concern, caused by the bacterium Neisseria gonorrhoeae. The main clinical feature of acute gonorrhea is neutrophilic influx that is unable to clear infection. Women of reproductive age are predominantly at risk for serious sequelae of gonorrhea, including pelvic inflammatory disease, ectopic pregnancy, and infertility.

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Background

Neisseria gonorrhoeae is the second most common bacterial cause of sexually transmitted infections (STIs) in North America, following Chlamydia trachomatis. Globally, gonococcal infections are now an urgent problem because N. gonorrhoeae is capable of rapidly developing resistance to multiple antibiotic classes. Over time, N. gonorrhoeae has become less susceptible to numerous antibiotics, including the sulfonamides, penicillins, tetracyclines and fluoroquinolones. More recently, cases of resistance to cephalosporins, the current first-line treatment, have been reported.

According to the Public Health Agency of Canada (PHAC), the incidence of gonorrhea has more than doubled, from approximately 15 cases per 100,000 in 1997 to up to 33 cases per 100,000 in 2009. In the United States in 2011, the reported rate was even higher, at 104.2 cases per 100,000. In both Canada and the United States, gonorrhea is more common in young adults (women aged 15-24 and men aged 20-24). In the United States, the occurrence in this age group is about 5 times that of the national average. The incidence of this infection, however, is confounded by factors such as changes in both reporting practices and screening, as well as the use of diagnostic tests with different sensitivities.

Risk factors for gonorrhea include sexual contact with an infected person or someone from an endemic area; previous gonorrhea, STIs or human immunodeficiency virus (HIV); being a sexually active youth; having multiple partners; and being a sex worker, street youth and/or man who has sex with men (MSM). Geographic clustering of gonococcal infections is associated with minority ethnic groups, low socioeconomic status and lack of education.

Gonorrhea is often asymptomatic in females and symptomatic in males. When symptomatic, the clinical presentation in females includes vaginal discharge, dysuria, dyspareunia, abnormal uterine bleeding, lower abdominal and/or rectal pain. In males, symptoms include urethral discharge and/or itch, dysuria and testicular or rectal pain. The urethra and cervix are the most frequently affected anatomical sites, followed by anal and pharyngeal areas.

Gonococcal infections are considered uncomplicated in the absence of bacteremia or pathogen spread to extragenital sites. However, left untreated, this infection can have serious sequelae. These include pelvic inflammatory disease, infertility, ectopic pregnancy and chronic pain in women and epididymo-orchitis, reactive arthritis and, rarely, infertility in men. In both genders, the infection can progress to disseminated disease. N. gonorrhoeae infection can also facilitate the transmission of HIV. Pharmacists can help their patients by being familiar with symptoms of gonorrhea infections and being aware of new guidelines and treatment regimens ().

Treatment

Considerations for selection of antimicrobial therapy

The ideal treatment regimen for gonorrhea should cure at least 95% of infections. In other words, an antibiotic to which more than 5% of N. gonorrhoeae strains exhibit resistance should not be used, given the close correlation between in vitro susceptibility testing and clinical failure. When treatment failures are minimized, the potential spread of resistant disease is also reduced. Also, antibiotic therapy should be effective at all anatomical sites, well tolerated and easy to comply with (preferably single-dose therapy at the point of care, i.e., directly observed therapy).

Summary of treatment guidelines

New recommendations for the treatment of gonorrhea have been published to address the increased prevalence of N. gonorrhoeae resistance. The Alberta Treatment Guidelines for STIs were updated in 2012, while the PHAC, Public Health Ontario and the Quebec Institut national d'excellence en santé et en services sociaux updated their guidelines in 2013. Updated 2014 guidelines are also available from British Columbia and Saskatchewan. Other provincial guidelines either have not yet been updated or are referring clinicians to the new Canadian guidelines.

The guidelines address the increasing resistance to cephalosporins, with recommendations varying based on the geographic region. Canadian guidelines recommend either intramuscular (IM) ceftriaxone or oral cefixime as the preferred antibiotic of choice. In contrast, the CDC and organizations in other countries advocate only parenteral cephalosporins as first-line therapy. Similar to the international guidelines, Ontario also recommends parenteral cephalosporins as the preferred regimen, given local reports of resistance to oral cefixime. The recommended dose of ceftriaxone IM varies between North America and other countries. Both Canada and the United States recommend the lower 250 mg dose, while Europe and the UK propose a higher dose of 500 mg.

All guidelines currently recommend co-treatment with azithromycin for C. trachomatis, regardless of chlamydia test results. The reasons for co-treatment include the high rate of coinfection and antigonococcal activity of azithromycin and doxycycline. The use of combination treatment for gonorrhea aims to improve treatment efficacy and delay emergence and spread of resistance to the cephalosporins.

Resistance patterns and mechanisms in the treatment of gonorrhea

Over the last 2 decades, new antimicrobial susceptibility surveillance programs such as Canada's National N. gonorrhoeae Surveillance Program were developed in response to the rapid rise of resistance to N. gonorrhoeae. This program is coordinated by the National Microbiology Laboratory from the PHAC. Each year the program reports rising numbers of gonorrhea cases, with an increasing proportion resistant to at least 1 antibiotic. Between 2000 and 2009, no reported isolates were resistant to either ceftriaxone or cefixime. However, a shift occurred in the modal minimum inhibitory concentration (MIC) for both drugs, including a combined total of 208 isolates with decreased susceptibility. Among these isolates, more exhibited reduced susceptibility to cefixime than to ceftriaxone. The mechanism for resistance was largely due to alterations in the penA, porB1b and mtrR genes, which diminish b-lactam binding to the cell wall, decrease permeability of cephalosporins and increase drug efflux from the cell, respectively.

In the United States, the Gonococcal Isolate Surveillance Project (GISP) monitors trends in antimicrobial susceptibilities of N. gonorrhoeae in order to guide treatment recommendations. GISP defines decreased susceptibility of N. gonorrhoeae to cefixime and ceftriaxone as an MIC of ≥0.5 mcg/mL. From 2008 to 2012, GISP reported a small increase in the percentage of N. gonorrhoeae isolates with an MIC ≥0.125 mcg/mL for ceftriaxone, from 0.1% to 0.3%. One isolate with an MIC ≥0.5 mcg/mL was found. A total of 4 isolates with decreased susceptibility (MIC of 0.5 mcg/mL) were reported between 1992 and 1997. For cefixime, the percentage of isolates ≥0.25 mcg/mL increased from 0.1% in 2006 to 1.0% in 2012. In 2012, 3 isolates resistant to cefixime were reported, 2 with MICs of 0.5 mcg/mL and 1 with an MIC of 1 mcg/mL.

Clinical treatment failures

Clinical cases of cephalosporin treatment failures in Canada are only recent. A retrospective cohort study published in 2013 from a Toronto sexual health clinic described treatment failure with cefixime. N. gonorrhoeae (with identical molecular typing to baseline) was isolated at the follow-up test-of-cure visit in 13 of 133 individuals. Nine individuals were reported to have failed cefixime in the treatment of urethral, rectal or pharyngeal gonococcal infections. The other 4 were not considered treatment failures as their records did not include information as to possible sexual reexposure. The clinical failure rate was 25% for those with a cefixime MIC of 0.12 mcg/mL or greater, compared with 1.9% for those with a cefixime MIC of less than 0.12 mcg/mL. In contrast, an Alberta study reported treatment failures in pharyngeal infections with cefixime 400 mg in 13.1% of patients, although failures were not related to elevated cefixime MICs but rather attributed to reduced drug concentrations of oral cephalosporins in the pharynx compared with other sites.

Prior to this study, most clinical failures were reported internationally. In Japan, a case of ceftriaxone-resistant N. gonorrhoeae (MIC of 2 mcg/mL) was reported in 2011 in a female commercial sex worker. In Sweden in 2010, a heterosexual male cultured positive more than once for ceftriaxone-resistant strains (MIC of 0.125 or 0.25 mcg/mL) requiring a 1 g dose of ceftriaxone for treatment success. In 2010, 3 cases of cefixime failure were reported, 2 in Norway and 1 in England, and all were in heterosexual men. In Austria in 2011, 1 case of cefixime failure was reported in an MSM. In all of these cases, the initial treatment was oral cefixime 400 mg. In most cases, ceftriaxone was prescribed following identification of the treatment failure.

Cefixime dosing

The use of the cephalosporins in the treatment of gonorrhea is a concern, regardless of whether ceftriaxone or cefixime at an elevated dose of 800 mg is used as first-line therapy. Note only does the lack of alternative therapies limit clinicians in tailoring therapies based on safety considerations such as drug allergies, pregnancy and adverse effects, but the focus on one class for therapy has historically led to a rapid rise in resistance. The newest guidelines address the issue of increasing resistance to the cephalosporins with the knowledge that choices are limited once this class is no longer effective.

The Canadian guidelines are unique, as they continue to recommend oral cefixime with an increased dose to overcome rising MICs. The 800 mg dose of cefixime is off-label use; however, the Canadian guidelines state that it is safe and effective and provides a prolonged time above the MIC when compared with the 400 mg dose. Data for the 800 mg dose of cefixime in patients with gonorrhea are limited to a few older trials.

A 1992 study compared both the 400 mg and 800 mg doses of cefixime with IM ceftriaxone 250 mg in patients with uncomplicated N. gonorrhoeae urethritis or cervicitis. Among the 155 evaluable patients, efficacy was comparable between the 3 groups, with bacterial eradication rates of 99%, 95% and 100%, respectively. In the 3 cases of treatment failure (1 case with cefixime 400 mg and 2 cases with cefixime 800 mg), the cefixime MIC ranged from 0.004 to 0.008 mcg/mL, while the geometric mean MIC for all 187 isolates was 0.005 mcg/mL. Three patients reported adverse effects after taking the 800 mg cefixime dose compared with 10 in the 400 mg group and none in the ceftriaxone group. The most frequent side effect reported with cefixime was diarrhea/loose stools (3% of the total group). A 1991 randomized unblinded study of 333 patients similarly evaluated oral cefixime 400 mg or 800 mg and ceftriaxone 250 mg IM. All 3 regimens again demonstrated similar rates of bacterial eradication (96%, 98% and 98%, respectively). All regimens were well tolerated, with 13% of patients reporting mild to moderate side effects. Gastrointestinal side effects (e.g., nausea, diarrhea and epigastric pain) occurred more frequently in the group taking 800 mg of cefixime compared with 400 mg of cefixime (18% vs. 8%). The 6 patients with persistent infection following cefixime therapy had baseline MICs of 0.004 mcg/mL ( n = 3) and 0.015 mcg/mL ( n = 3). The 2 patients with persistent infection following ceftriaxone therapy had baseline MICs of 0.001 and 0.004 mcg/mL.

Approach to treatment failures/prevention of spread and resistance

The PHAC recommends consultation with infectious disease specialists along with culture and sensitivity testing for cases of treatment failure. Therapy includes higher doses of ceftriaxone ranging from 250 mg to 1 g IM given in combination with azithromycin 1 g orally. Second-line therapy and treatment failure therapy all require a test of cure (see ). It is essential that the development of resistance to N. gonorrhoeae be minimized by optimizing treatment options based on geographical resistance patterns. Patient education and partner notification are of utmost importance in improving public awareness and reducing the spread of this resistant organism ().

Conclusion

The growing resistance of N. gonorrhoeae to cephalosporins has led to the use of increased doses of ceftriaxone and cefixime to ensure effective treatment. The PHAC continues to recommend oral cefixime but at an increased dose of 800 mg, or IM ceftriaxone, with only the latter recommended for MSM and pharyngeal infections. These recommendations are based on geographical differences of MICs across the country, while allowing for provincial/territorial differences in guidelines. Guidelines from the province of Ontario, the United States and Europe endorse IM ceftriaxone as the sole first-line agent for all gonococcal infections. Of note, Sanofi-Pasteur has recently announced a nationwide shortage of cefixime predicted to last until October 2015. Although provincial public health agencies are reserving cefixime specifically for the treatment of gonococcal infections, supplies may not last until the end of the back order. N. gonorrhoeae has demonstrated rapid changes in its susceptibility patterns over the years. In response, the public health agencies remain vigilant in monitoring the evolution of this organism and maintaining up-to-date guidelines. Pharmacists can play a role by educating their patients with respect to compliance, follow-up, partner notification and strategies for prevention of transmission of this ever-adaptable organism. ■

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Gonorrhea: Treatment update for an increasingly resistant organism

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Neisseria gonorrhoeae is the second most common bacterial cause of sexually transmitted infections (STIs) in North America, following Chlamydia trachomatis. Globally, gonococcal infections are now an urgent problem because N. gonorrhoeae is capable of rapidly developing resistance to multiple antibiotic classes. Over time, N.

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Biological feasibility and importance of a gonorrhea vaccine for global public health

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Biological feasibility and importance of a gonorrhea vaccine for global public health

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Highlights

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78 million new infections annually; greatest impact on women and neonates in LMIC.

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Current control measures are inadequate and challenged by antibiotic resistance.

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Conserved candidate vaccine antigens and adjuvant strategies are being developed.

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There is a need for human studies to investigate correlates of immunity.

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A meningococcal outer membrane vesicle vaccine may protect against gonorrhea.

Abstract

There is a growing public health interest in controlling sexually transmitted infections (STIs) through vaccination due to increasing recognition of the global disease burden of STIs and the role of STIs in women's reproductive health, adverse pregnancy outcomes, and the health and well-being of neonates. Neisseria gonorrhoeae has historically challenged vaccine development through the expression of phase and antigenically variable surface molecules and its capacity to cause repeated infections without inducing protective immunity. An estimated 78 million new N. gonorrhoeae infections occur annually and the greatest disease burden is carried by low- and middle-income countries (LMIC). Current control measures are clearly inadequate and threatened by the rapid emergence of antibiotic resistance. The gonococcus now holds the status of "super-bug" as there is currently no single reliable monotherapy for empirical treatment of gonorrhea. The problem of antibiotic resistance has elevated treatment costs and necessitated the establishment of large surveillance programs to track the spread of resistant strains. Here we review the need for a gonorrhea vaccine with respect to global disease burden and related socioeconomic and treatment costs, with an emphasis on the impact of gonorrhea on women and newborns. We also highlight the challenge of estimating the impact of a gonorrhea vaccine due to the need for more data on the burden of gonococcal pelvic inflammatory disease and related sequelae and of gonorrhea-associated adverse pregnancy outcomes and the problem of empirical diagnosis and treatment of STIs in LMIC. There is also a lack of clinical and basic science research in the area of gonococcal/chlamydia coinfection, which occurs in a high percentage of individuals with gonorrhea and should be considered when testing the efficacy of gonorrhea vaccines. Finally, we review recent research that suggests a gonorrhea vaccine is feasible and discuss challenges and research gaps in gonorrhea vaccine development.

Keywords

Pelvic inflammatory disease

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# data[].attributes.attachment_link.title
Biological feasibility and importance of a gonorrhea vaccine for global public health

# data[].attributes.attachment_link.description
JavaScript is disabled on your browser. Please enable JavaScript to use all the features on this page. * 78 million new infections annually; greatest impact on women and neonates in LMIC. * Current control measures are inadequate and challenged by antibiotic resistance. * Conserved candidate vaccine antigens and adjuvant strategies are being developed.