Course:PATH417:2022W2/Case2

From UBC Wiki

Case: 21-year-old Naser recently hooked up with a new sexual partner. This morning he noticed a burning pain in his urethra during urination, followed by a yellow-white discharge. He decided to visit the student health clinic where the doctor asks Naser about his recent sexual history. He recounts that he had unprotected sexual intercourse with a new partner about one week ago. When asked, the new partner claims no knowledge of having any sexually transmitted infections. The doctor asks Naser to provide a urine sample to send to the Microbiology Laboratory. The laboratory report results positive for Neisseria gonorrhoeae. The doctor prescribes antibiotics for Naser and counsels him on safe sex practices and on the importance of encouraging his new partner to come in for testing too. Naser is advised that he will be contacted by public health to discuss any other sexual partners he might have.


1. The Body System

(i) Describe the signs (objective characteristics usually detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient). Are there any other signs or symptoms that could have been commented on but are not presented in the case? What are the key History of Presenting Illness elements presented? What laboratory samples are taken and why? What are the meanings of the laboratory results reported? (No need to describe physiology of the signs and symptoms and no need to describe the laboratory testing itself as these are the basis of other questions).

Key signs and symptoms of Neisseria Gonorrhea

Neisseria Gonorrhea (N. gonorrhea) is a sexually transmitted disease (STD) that occurs as an infection that involves the reproductive organs of both males and females (1). It does not always have symptoms, particular in the case of women. Since in women, gonococcal inflammation does not occur at the same location as urination does and is less painful, this makes symptoms more difficult to notice, or the symptoms could be mild (2,3,4). Common symptoms of gonorrhea in both men and women include dysuria or pain, burning/stinging sensation, and itch in the urethra during urination, itchy or sore throat and pain in the anus plus discharge during bowel movement (2,3,5). Men may also experience signs such as white, yellow or green discharge from the penis and testicular pain (2,3,5). Symptoms that women may also experience include lower abdominal pain, dyspareunia (painful sexual intercourse), white or yellow vaginal discharge, abnormal vaginal bleeding, e.g. bleeding between periods (2,3,5), and bartholinitis, which is the infection and inflammation of the Bartholini’s glands (6). Swollen testicles and Bartholin’s cyst are visible and are, therefore, signs that can be observed by healthcare professionals (2,6). Eyelid edema, conjunctival erythrema, and profuse purulent discharge are signs of ophthalmia neonatorum or eye infection in infants born to women with gonococcal infection in their vaginas (5,6,7).

The signs and symptoms for women and newborn infants are not presented in the case, obviously because they do not apply to Naser’s case. Also, testicular pain, swollen testicles, and symptoms associated with gonococcal pharyngeal infection in this case of people who engage in oral sex partners who have gonococcal infection (8,9), and anorectal gonococcal infection in the case of people engaging in unprotected sex with partners with gonorrhea are not mentioned in this case (10). Perhaps that is because Naser did not experience these symptoms and, therefore, did not mention either symptom to the doctor.

History of present illness

The key elements of history of present illness are location, quality, severity, duration, timing, context, modifying factors, and associated signs and symptoms. Naser noticed the pain in his urethra and the yellow-white discharge from his penis while he was urinating in the morning. He went to the student health clinic later in the day. Naser experienced the burning pain and discharge when urinating in the morning. He might have experienced the symptoms again before he went to the clinic but the case doesn’t make any mention. Naser had unprotected sexual intercourse with a new partner about one week before he experienced the symptoms. Naser mentioned to the doctor that he had asked and the new partner claimed to have no knowledge of having any sexually transmitted disease. The symptoms of gonococcal infection usually present 2-14 days after sexual intercourse (11). Therefore, if Naser didn’t have any unprotected sex more than a week before he had sex with the new partner, it is likely that he contracted gonorrhea from the new partner. The symptoms are burning pain in the urethra during urination followed by yellow-white discharge from the penis. The doctor at the clinic could not make any observation of any signs.

Meanings of the laboratory results reported

A urine sample from Naser was taken and sent to the Microbiology Laboratory for analysis. This was to allow laboratory testing to be done to confirm whether Naser did have N. gonorrhea, as suggested by his symptoms. Most likely, a nucleic acid amplification test (NAAT) was conducted on the urine sample (12). NAAT is the preferred diagnostic test for N. gonorrhea because of the simplicity of the sampling procedure and and the high accuracy of the diagnostic test (12). Taking a urine sample is non-invasive and easy. The sample provider just needs to refrain from urinating or voiding for 2 hours prior to sample collection. He/she then proceeds to collect the initial 10-20 ml of the urine stream without having to cleanse the genital area first (12,13). NAATs work by looking for genetic material, ie. DNA, of N. gonorrhea and are considered to be the most sensitive type of diagnostic tests for this bacterium (12).

The positive results for N. gonorrhea confirmed that Naser had gonococcal urogenital infection, as was suggested by his symptoms, and that it is likely that N. gonorrhea bacteria were passed through genital contact from his new partner as they had unprotected sexual intercourse a week earlier.

References

1. McSheffrey GG, Gray-Owen SD. 2015. Chapter 82, Neisseria gonorrhoeae. In Tang YW, Sussman M,  Liu DY, Poxton I, Schwartzman J(ed), Molecular Medical Microbiology, Second Edition (pp. 1471-1485). Academic Press. http://dx.doi.org/10.1016/b978-0-12-397169-2.00082-2

2. Gonorrhea: Causes, Symptoms, Treatment & Prevention. Cleveland Clinic. Last reviewed September 2, 2022. Accessed February 9, 2023. https://my.clevelandclinic.org/health/diseases/4217-gonorrhea

3. Gonorrhea – CDC Detailed Fact Sheet. Centers for Disease Control and Prevention. Last Reviewed December 1, 2022. Accessed February 9, 2023. https://www.cdc.gov/std/gonorrhea/stdfact-gonorrhea-detailed.htm

4. Quillin SJ, Seifert HS. 2018. Neisseria gonorrhoeae host-adaptation and pathogenesis. Nature reviews Microbiology. 16(4). doi:10.1038/nrmicro.2017.169

5. Gonorrhea guide: Risk factors and clinical manifestations - Canada.ca. Public Health Agency of Canada. Last modified April 28, 2022. Accessed February 9, 2023. https://www.canada.ca/en/public-health/services/infectious-diseases/sexual-health-sexually-transmitted-infections/canadian-guidelines/gonorrhea/risk-factors-clinical-manifestation.html

6. Ghanem K. 2022. Marrazzo, J., & Bloom, A, (Eds.). Clinical manifestations and diagnosis of Neisseria gonorrhoeae infection in adults and adolescents. UpToDate. https://www.uptodate.com/contents/clinical-manifestations-and-diagnosis-of-neisseria-gonorrhoeae-infection-in-adults-and-adolescents#H790871163

7. Ophthalmia Neonatorum, Clinical Management Guidelines. The College of Optometrists. Retrieved from https://www.college-optometrists.org/clinical-guidance/clinical-management-guidelines/ophthalmianeonatorum

8. Bro-Jorgensen A, Jensen T. 1973. Gonococcal pharyngeal infections. Report of 110 cases. Sexually Transmitted Infections. 49(6):491-499. doi:10.1136/sti.49.6.491

9. Silber TJ, Controni G. 1983. Clinical spectrum of pharyngeal gonorrhea in children and adolescents. Journal of Adolescent Health Care. 1983;4(1):51-54. doi:10.1016/s0197-0070(83)80230-7

10. Klein EJ, Fisher LS, Chow AW, Guze LB. 1977. Anorectal Gonococcal Infection. Annals of Internal Medicine. doi/10.7326/0003-4819-86-3-340

11. What Are the Symptoms & Signs of Gonorrhea? Planned Parenthood. Accessed February 9, 2023. https://www.plannedparenthood.org/learn/stds-hiv-safer-sex/gonorrhea/what-are-symptoms-gonorrhea

12. Gonorrhea guide: Screening and diagnostic testing - Canada.ca. Public Health Agency of Canada. Last modified April 28, 2022. Accessed February 9, 2023.

(ii) Which body systems are affected in this case and what other anatomical sites could be affected by this infection? In what way has the normal physiological functioning (in the described case and potential other infection sites or for female patients or other specific patient populations) of this body system been disturbed by the infection (specifically looking at the physiological changes without detailing the bacterial mechanism of this disturbance as that is the basis of another question). Representing this diagrammatically is helpful to demonstrate understanding.

In the case study, only the urethra is affected, as indicated by the painful sensation during urination and the presence of urethral discharge. However, other anatomical sites that could be affected include the joints due to gonococcal arthritis, the rectum due to proctitis, the conjunctiva due to conjunctivitis, and the pharynx due to pharyngitis (1). In women specifically, the pelvic area may also be affected due to the pathogen spreading to the uterus and fallopian tubes, causing pelvic inflammatory disease (PID), which can cause infertility (1). 45% of women experience ascending infection from the lower to upper female genital tract after cervical epithelial cells infection (2). Gonococci release of phospholipase D modulates host signal transduction in cervical epithelial cell to promote CR3 recruitment to the cell surface (3). The binding of Gonococcal pilus to the CR3 causes actin remodelling and membrane ruffling that facilitates host cell entry in macropinosomes (3). Gonococci can also inactivate the complement system to enhance extracellular survival within the lower female genital tract (4). In-vivo studies demonstrate that sialyated lipooligosaccharide (LOS) enhances factor H deposition, leading to the degradation of C3 in the alternative complement pathway (4). Furthermore, Gonococcal peptidoglycan and LOS simulates the production of TNF cytokines, leading to the shedding of ciliated fallopian tube epithelial cells (5). The loss of ciliated cells allows gonococci to be transcyotsed to the basolateral surface and infection of subepithelial tissues, where dissemination into the bloodstream can occur (5). The disseminated infection leads to complications, including arthritis, dermatitis and vasculitis (4). If a pregnant women is left untreated, it can result in chorioamnitis, the fetal membranes to prematurely rupture, and premature birth of a fetus with low weight (6). Furthermore, the infection can be passed onto the newborn during delivery, resulting in conjunctivitis of the neonate (6). In males, gonoccoi infection of urethral epithelial cells elicits an inflammatory response through L-8, IL-6, IL-1β and TNF-α secretion (1). This triggers polymorphonuclear neutrophils (PMN) recruitment and shedding of urethral epithelial cells, which is associated with urethral discharge (2). The insertion of porin protein into the PMN membrane modulates PMN host cell response including releasing toxic oxidants into the extracellular environment (7). Porin also inhibits degranulation, phagosome maturation and expression of cell surface receptors for immune function in PMN (8). This mechanism can induce penile inflamation, causing the patient to experience pain in the area (1). Moreover, in the case study, only the normal physiological function of the urethra has been affected. However, in addition to the urethra, physiological functions such as feces excretion may be affected due to a rectal infection, eating or speaking abilities may be affected due to pharyngitis causing a sore throat, joint movement may be difficult or painful due to gonococcal arthritis, and sex may be painful due to vaginal irritation in women or urethral infection in men (1).

References:

  1. Springer, C., & Salen, P. (2022, April 21). Gonorrhea - StatPearls - NCBI Bookshelf. Gonorrhea. Retrieved February 10, 2023, from https://www.ncbi.nlm.nih.gov/books/NBK558903/
  2. Harvey HA, Ketterer MR, Preston A, Lubaroff D, Williams R, Apicella MA. Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria Gonorrhoeae. Infection and Immunity. 1997;65(6):2420–7.
  3. Haines KA, Yeh L, Blake MS, Cristello P, Korchak H, Weissmann G. Protein I, a translocatable ion channel from neisseria gonorrhoeae, selectively inhibits exocytosis from human neutrophils without inhibiting O2- generation. Journal of Biological Chemistry. 1988;263(2):945–51.
  4. Mosleh IM, Huber LA, Steinlein P, Pasquali C, Günther D, Meyer TF. Neisseria gonorrhoeae porin modulates phagosome maturation. Journal of Biological Chemistry. 1998;273(52):35332–8.
  5. Edwards JL, Entz DD, Apicella MA. Gonococcal phospholipase D modulates the expression and function of complement receptor 3 in primary cervical epithelial cells. Infection and Immunity. 2003;71(11):6381–91.
  6. 2022. Detailed std facts - stds & pregnancy. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention.
  7. Whiley DM, Tapsall JW, Sloots TP. Nucleic acid amplification testing for neisseria gonorrhoeae. The Journal of Molecular Diagnostics. 2006;8(1):3–15.
  8. Harvey HA, Post DM, Apicella MA. Immortalization of human urethral epithelial cells: A model for the study of the pathogenesis of and the inflammatory cytokine response to neisseria gonorrhoeae infection. Infection and Immunity. 2002;70(10):5808–15.

(iii) What antibiotics/treatments might have been given (i.e., what are antibacterial treatments and how do these treatments work to help the body clear the organism)? Are there any considerations for antimicrobial resistance for this organism? Representing this diagrammatically is helpful to demonstrate understanding.

Antibacterial treatment options for N. gonorrhoeae infection

For the treatment of uncomplicated urethritis, the recommended treatment regimen is described in table 1. Traditional treatment regimen for gonorrhea used to be dual therapy using ceftriaxone and azithromycin. (1, 2. 3). Ceftriaxone is a third-generation cephalosporin antibiotic that covers gram-negative broadly. (4) Ceftriaxone kills bacteria by inhibiting bacterial wall synthesis and N. gonorrhoeae is susceptible to ceftriaxone. (4) Azithromycin is a macrolide antibiotic that works by inhibiting bacterial protein synthesis. (5) Azithromycin is indicated for N. gonorrhoeae infection. (5) Treatment options for PID, acute epididymitis, and disseminated gonococcal infection are described in table 2.

Table 1. The recommended regimen of uncomplicated gonococcal urogenital infection (1, 2, 3, 6)

Table 2. The recommended regimen of gonococcal infection for PID, acute epididymitis, and disseminated gonococcal infection (1)

  • Tables for the treatment options of gonorrhea.png

Antimicrobial resistance for N. gonorrhoeae

There are concerns about the increasing resistance against macrolide as minimum inhibitory concentrations (MICs) of antibiotics have been increasing each year until 2016. (1, 3) Because of the resistance, azithromycin is removed from new STI guidelines from CDC recommends intramuscular ceftriaxone 500 mg single dose. (3, 6)

References

  1. Bennett JE, Dolin R, Blaser MJ. Chapter 212. Neisseria gonorrhoeae (Gonorrhea). In: Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. Philadelphia, PA: Elsevier; 2020.
  2. British Columbia Treatment Guidelines: Sexually Transmitted Infections in Adolescents and Adults (2014) BC Centre for Disease Control. Available at: http://www.bccdc.ca/resource-gallery/Documents/Communicable-Disease-Manual/Chapter%205%20-%20STI/CPS_BC_STI_Treatment_Guidelines_20112014.pdf (Accessed: February 9, 2023)
  3. DiPiro JT. Chapter 18: Infectious Disease. In: Pharmacotherapy: A Pathophysiologic Approach. New York: McGraw Hill; 2020.
  4. Ceftriaxone. Uses, Interactions, Mechanism of Action | DrugBank Online. https://go.drugbank.com/drugs/DB01212. Accessed February 10, 2023.
  5. Azithromycin. Uses, Interactions, Mechanism of Action | DrugBank Online. https://go.drugbank.com/drugs/DB00207. Accessed February 10, 2023.
  6. Gonococcal infections among adolescents and adults - STI treatment guidelines (2022) Centers for Disease Control and Prevention. Centers for Disease Control and Prevention. Available at: https://www.cdc.gov/std/treatment-guidelines/gonorrhea-adults.htm (Accessed: February 9, 2023).

(iv) Why did the doctor discuss sexual partners and their notification? Is this a reportable communicable disease? What are the recommendations for follow up of partners? Should any other testing be done for Nasir? What are the recommendations for safe sex practices for someone with N. gonorrhoeae infection? Now that Nasir had N. gonorrhoeae once, does it mean that he shouldn’t worry about getting it again?

Granted Gonorrhea is a sexually transmitted infection, the Doctor in this case wished to discuss Naser’s past sexual partners in order to determine who transmitted the infection to him, and (hopefully) stop the infection from being spread further. Naser was advised that public health would be in contact with him to discuss sexual partnerships in order to seek the partner that transmitted the disease, have them get treated, and therefore minimize the spread of this bacterial infection. Generally speaking, Gonorrhea is a reportable communicable disease which is heavily dependent on individual’s ability to report their positive cases to their sexual partners considering the reporting requirements vary depending on local government guidelines (1). It is highly recommended to follow up with sexual partners after testing positive for Gonorrhea as they may also be infected and thus require treatment - regardless if they are showing symptoms or not (2). If there is no communication with past partners and they are left untreated, Gonorrhea could lead to very serious health issues and may spread to other people during future sexual encounters (2).

In order to rule out other possible co-existing infections, health professionals advise additional STI testing (3). As an example, those who test positive for Gonorrhea are at an increased risk for co-infection with Chlamydia and Syphilis (3). In addition, routine testing for other blood borne pathogens such as HIV is considered standard practice granted the increased risk that Gonorrhea has on both acquiring and transmitting HIV (3). Moreover, public health officials advise refraining from sexual activity for a minimum of seven days after starting the first dose of antibiotic treatment as long as all symptoms have subsided – same goes for sexual partners on antibiotic treatment (4). Re-testing for Gonorrhea is critical three months post primary infection to ensure all of the initial infection has successfully cleared, and no re-infection has occurred (5). Although permanent damage caused from the disease will never heal, with the correct treatment using antibiotics, Gonorrhea can be cured (5). That being said, after having Gonorrhea once, re-infection can occur by having sexual intercourse with someone positive for Gonorrhea; therefore, Naser – after already having this STI – should remain aware, vigilant, and embrace a safe sex lifestyle in order to minimize his chance of becoming re-infected with N. gonorrhoeae (5).


References:

1. CDC, C. D. C. (2021, July 22). Reporting and confidentiality. Centers for Disease Control and Prevention. Retrieved February 10, 2023, from https://www.cdc.gov/std/treatment-guidelines/clinical-reporting.htm

2. CDC, C. D. C. (2020, March 31). Just diagnosed? next steps after testing positive for gonorrhea or Chlamydia. Centers for Disease Control and Prevention. Retrieved February 10, 2023, from https://www.cdc.gov/std/prevention/NextSteps-GonorrheaOrChlamydia.htm

3. Canada, P. H. A. of. (2022, April 28). Government of Canada. Canada.ca. Retrieved February 10, 2023, from https://www.canada.ca/en/public-health/services/infectious-diseases/sexual-health-sexually-transmitted-infections/canadian-guidelines/gonorrhea/screening-diagnostic-testing.html

4. Springer, C., & Salen, P. (2022). Gonorrhea - StatPearls - NCBI Bookshelf. National Library of Medicine. Retrieved February 11, 2023, from https://www.ncbi.nlm.nih.gov/books/NBK558903/

5. CDC, C. D. C. (2022, August 22). Std facts - gonorrhea. Centers for Disease Control and Prevention. Retrieved February 10, 2023, from https://www.cdc.gov/std/gonorrhea/stdfact-gonorrhea.htm



2. The Microbiology Laboratory

(i) What are the most common pathogens associated with urethritis?

The three most common pathogens associated with urethritis are Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium (1). Herpes simplex types 1, type 2, and adenovirus are viral pathogens that can cause urethritis but are less common than bacterial infections (1). Regarding this case, Naser tested positive for N. gonorrhoeae.

Neisseria gonorrhoeae

N. gonorrhoeae is a leading cause of urethritis in both men and women along with C. trachomatis (2). Pain during urination and discharge from genitals are some of the common symptoms of urethritis (3, 4). Infected men present with purulent urethritis, discharge and dysuria (5). Whereas, infected women may experience urethritis, cervical discharge and Pelvic inflammatory Disease (PID; 5). However, many women are observed to be less symptomatic than men (5). N. gonorrhoeae may also cause ascending infections such as epididymitis and salpingitis in men (3). The majority of N. gonorrhoeae infections are local and disseminated infections such as arthritis, endocarditis or vasculitis are rare (4, 3).

Additionally, N. gonorrhoeae infections caused by unprotected sexual activity could affect the genitals, rectum, and pharynx (4). Moreover, N. gonorrhoeae transmission can be congenital which could infect conjunctiva of newborns during birth if their mother was infected with this pathogen (4).

Neisseria gonorrhoeae is a gram-negative diplococcus with flat adjacent sides acquired through sexual contact. N. gonorrhoeae adheres to columnar epithelial cells, then penetrates and multiplies in the basement membrane of the infected mucosal epithelium of urogenital tracts, the rectum, pharynx, and/or the conjunctiva (3, 6). Gonococcal lipopolysaccharides stimulate the production of tumor necrosis factor causing tissue damage (6).

This sexually transmitted disease is prevalent all over the world and host-related factors include the number of sexual partners, safe sex practices and sexual preferences (6).

Chlamydia trachomatis

Chlamydia trachomatis is the most common bacterial sexually transmitted infection in the world (7, 8) and is extremely problematic in many fields of medicine including ophthalmology, dermatology, and rheumatology (8). The incidence of Chlamydia trachomatis is over 90 million people per year according to the World Health Organization (8). Unfortunately, Chlamydia trachomatis infections are commonly undiagnosed making the actual rate of incidence higher than what is reported (8).

Untreated infections may lead to serious complications including reactive arthritis, commonly referred to as Reiter’s syndrome (2, 9). In men, C. trachomatis may lead to epididymitis and orchitis (9). Women may experience pelvic inflammatory disease (PID), ectopic pregnancy, infertility and more (9). Many infected patients are asymptomatic despite the possible serious complications (9). Most cases of urethritis caused by C. trachomatis are asymptomatic, but in symptomatic cases, dysuria and urethral discharge can occur.

Mycoplasma genitalium

M. genitalium is also a common pathogen associated with urethritis, and it is a self-replicating bacteria without a cell wall (1). These infections are also commonly asymptomatic, but can cause symptoms of dysuria, purulent or mucopurulent urethral discharge, as well as urethral pruritus, balanitis, and posthitis (10). It is commonly found in co-infection with both N. gonorrhoeae and Mycoplasma genitalium, other possible causes of urethritis (10)

M. genitalium is flask-shaped with a curved terminal organelle and is very small, measuring 0.2μm to 0.7μm. It is also slow growing and possesses a small genome of only 521 genes total and 482 encoding proteins (11). Today, there is no gold standard for laboratory testing for M. genitalium due to the lack of standardization and how difficult it is to culture (11, 12). Due to this bacterium’s slow growth, nucleic acid amplification tests are the best way to diagnose urethritis related to M. genitalium (12). Future studies must focus on the prevalence and risk of infection to further our understanding of this bacteria (11).


References:

  1. Bachmann LH, Manhart LE, Martin DH, Seña AC, Dimitrakoff J, Jensen JS, Gaydos CA. 2015. Advances in the Understanding and Treatment of Male Urethritis. Clin Infect Dis 61:S763–S769.
  2. Brill JR. Diagnosis and treatment of urethritis in men. Am Fam Physician. 2010 Apr 1;81(7):873-8.
  3. Unemo M, Seifert HS, Hook EW 3rd, Hawkes S, Ndowa F, Dillon JR. Gonorrhoea. Nat Rev Dis Primers. 2019 Nov 21;5(1):79. doi: 10.1038/s41572-019-0128-6.
  4. Sadoghi B, Kränke B, Komericki P, Hutterer G. 2022. Sexually transmitted pathogens causing urethritis: A mini-review and proposal of a clinically based diagnostic and therapeutic algorithm. Front Med (Lausanne) 9:931765.
  5. Mayor MT, Roett MA, Uduhiri KA. Diagnosis and management of gonococcal infections. Am Fam Physician. 2012 Nov 15;86(10):931-8. Erratum in: Am Fam Physician. 2013 Feb 1;87(3):163.
  6. Morse SA. 1996. Neisseria, Moraxella, Kingella and Eikenella, In Baron, S (ed.), Medical Microbiology, 4th ed. University of Texas Medical Branch at Galveston, Galveston (TX).
  7. Bennet JE, Dolin R, Blaser MJ. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 9th Ed. Philadelphia, PA: Elsevier; 2020.
  8. Choroszy-Król I, Frej-Mądrzak M, Jama-Kmiecik A, Bober T, Sarowska J. 2012. Characteristics of the Chlamydia trachomatis species – Immunopathology and Infections. Adv Clin Exp Med. 21(6):799-808.
  9. Mishori R, McClaskey EL, WinklerPrins VJ. Chlamydia trachomatis infections: screening, diagnosis, and management. Am Fam Physician. 2012 Dec 15;86(12):1127-32.
  10. Young A, Toncar A, Wray AA. 1 December 2022. Urethritis. In StatPearls [Internet]. StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK537282/. Retrieved 7 February 2023.
  11. Daley G, Russell D, Tabrizi S, McBride J. 2014. Mycoplasma genitalium: a review. Int J STD AIDS 25:475–487.
  12. Sethi S, Singh G, Samanta P, Sharma M. 2012. Mycoplasma genitalium: An emerging sexually transmitted pathogen. Indian J Med Res 136:942–955.


(ii) What samples should be taken for diagnostic testing?

The following samples are analyzed in the laboratory:

Urethral swab: Swab sampling is carried out by inserting a water-moistened swab with a rayon or Dacron tip 2-3 centimeters into the male urethra and gently rotating 2-3 times. It is preferable that the individual not have voided in the preceding 2 hours. Exudate collection is adequate to allow N. gonorrheae culture and smear microscopy (1,2). These specimens should be transported to a gram-stain plate immediately and rolled gently to preserve cellular morphology (2).

Urine sample: The initial 10 - 20 mL of urine stream, i.e., first-void urine sample is collected in a leak-proof container in order to detect shed organisms. While it is preferable that the individual to not have voided in the 2 hours preceding specimen collection, this does not preclude testing (7). First-void urine is the recommended sample type as it is non-invasive compared to other types, and is thus preferred over urethral swabs for pathogen detection, especially using NAATs (1,2,3,4).

Other populations: The aforedescribed specimens are collected from male patients presenting with urethritis, as in Naser’s case. Among MSM (men who has sex with men), infection with N. gonorrheae is localized at extragenital sites. As such, specimen collection from all sites of possible exposure is recommended (5). Genital infection with N. gonorheae tends to present with vaginal discharge among women in whom the infection tends to be asymptomatic (6). As such, vaginal swabs are recommended in this population. Cervical swabs may also facilitate diagnosis among women (2,5).

References

  1. Centres for Disease Control and Prevention (CDC). Recommendations for the Laboratory-Based Detection of Chlamydia trachomatis and Neisseria gonorrhoeae — 2014. MMWR 2014;63(No. RR-2).
  2. Ng LK, Martin IE. 2005. The laboratory diagnosis of Neisseria gonorrhoeae. Can J Infect Dis Med Microbiol 16:15–25.
  3. Public Health Agency of Canada (PHAC). Gonorrhea guide: Screening and diagnostic testing. Ottawa, ON: Government of Canada; 2022 Apr; 2022. Available from: https://www.canada.ca/en/public-health/services/infectious-diseases/sexual-health-sexually-transmitted-infections/canadian-guidelines/gonorrhea/screening-diagnostic-testing.html
  4. Bachmann LH, Manhart LE, Martin DH, Seña AC, Dimitrakoff J, Jensen JS, Gaydos CA. 2015. Advances in the Understanding and Treatment of Male Urethritis. Clin Infect Dis 61:S763–S769.
  5. Tuddenham S, Hamill MM, Ghanem KG. Diagnosis and Treatment of Sexually Transmitted Infections: A Review. 2022. JAMA 2022;327(2):161-172.
  6. Unemo M, Seifert HS, Hook III EW, Hawkes S, Ndowa F, Dillon JA. Gonorrhoea. Nature Reviews Disease Primers. 2019 Nov 21;5(1):79.
  7. Leos-Alvarado C, Llaca-Díaz J, Flores-Aréchiga A, Pérez-Chávez F, Casillas-Vega N. Male urethritis. A review of the ideal diagnostic method. Actas Urológicas Españolas (English Edition). 2020 Oct 1;44(8):523-8.

(iii) Explain the tests that can be performed on samples to detect any of the potential bacterial pathogens associated with this disease. What are the advantages and disadvantages of these tests?

Microscopy: There are two broad classifications of bacteria depending on their cell membrane composition (1). Gram-negative bacteria present with thin layers of peptidoglycan (PG) and contain lipopolysaccharides (LPS) on their outer membrane. Whereas, gram-positive bacteria consist of thick layers of PG and there is no presence of the outer membrane with LPS (1). In order to distinguish between gram-negative and gram-positive bacteria under microscopy, a laboratory technique called gram-staining is performed (1). During gram-staining, a chemical complex called violet-crystal is used to detect the presence of PG layers (1). The greater amount of violet-crystal is bound to the gram-positive bacteria with a thick PG layer (1). During the ethanol decolorization step, violet-crystal compounds bound in gram-positive bacteria are washed less by ethanol compared to the gram-negative bacteria with a thin layer of PG (1). As a result, gram-positive bacteria depict purple colouration and gram-negative bacteria show pink colouration under microscopy (1).

N. gonorrhoeae is a gram-negative intracellular bacterium that contains a thin layer of PG and contains LPS on its cell wall (1, 2). Thus, the gram-staining results would show pink or red colouration (2). When using high magnification (1000x), more detailed patterns associated with N. gonorrhoeae can be observed which include the diplococci structure of the bacteria (2). The typical morphological pattern found in N. gonorrhoeae are diplococci structures that are perpendicular to each other and kidney-shaped (2).

The microscopy technique allows for the classification of the bacteria into broad categories which may be helpful in identifying the possible infectious agents causing urethritis. However, further advanced testing is required as many bacteria in the same species share similar cell wall components and morphology (2). Moreover, sample choice is important to avoid false diagnostic results (3). For example, rectal and oropharyngeal samples are not recommended as there is the presence of other gram-negative cocci pathogens that reduce sensitivity (3).

Bacterial Culture: Culture was the first line diagnostic test before the emergence of NAAT, and the costs are lower (3). Urethral swabs (for males) and endocervical swabs (for females) are most proper samples in detection of N. gonorrhoeae through culture (3, 5). Urethral samples can present a sensitivity level of 72-95% and improve it to 95-100% by more accurate performance (collection and transferring of samples) (3). Gonococcal species could show resistance to some of the classes of antibiotics (cephalosporins and macrolides). As a result, culture is the main test in recognizing antibiotic resistance and provides the most effective treatment for the target pathogen (5). Culture is recommended when the previous therapy failed to eradicate bacteria and there is a suspicion of pelvic inflammatory disease. Moreover, culture is the proper test in the cases of sexual assault victims (3, 5, 6). Patients with extragenital symptoms could provide rectal or oropharyngeal samples for culture if NAAT is not available; however, the sensitivity of these samples are lower compared to urogenital samples for the detection of gonorrhea (3, 5). If samples for culture are received < 48 hours, it might present negative results. As a result, for the confirmation of diagnosis another test might be required in a few weeks (3). Culture performance requires proper collection of samples and transferring as bacteria need to be alive (6). The environment should be rich with CO2 and temperature sets on 37 ºC (5). Selective and non-selective mediums are used for the culture (6). For the detection of  N. gonorrhoeae Thayer Martin agar, which is a selective medium with antimicrobial agents, is preferred to prevent the growth of other possible species (5, 6). Moreover, the type of swabs, like plastic and calcium alginate, are another key factor during collection of the samples to not damage N. gonorrhoeae species (5).  

Nucleic Acid Amplification Tests (NAATs): The nucleic acid amplification test (NAAT) is the gold standard method for diagnosing N. gonorrhoeae (7). It is reported that NAATs have the highest sensitivity and specificity among other diagnostic tests such as culturing (8). In high-income countries, NAATs are becoming a more favourable method of diagnosing in which the sample collection is non-invasive, i.e. urine samples (3). The use of bacterial cultures was the standard for N. gonorrhoeae testing, but the process of collecting cervical or urethral sample specimens was considered an invasive procedure (3). In contrast, NAATs generate highly sensitive and specific results from urine samples (3). Previously, urine samples were not recommended for N. gonorrhoeae diagnostic testing because the samples were not successfully cultivated (2). NAATs do not require transporting and maintaining viable organisms, which allows for more simple and effective testing in laboratory conditions (7, 8). There are many other advantages which include the generation of rapid results, inexpensive price, and relatively simple technique to perform in the laboratory (8). It is reported that NAATs have over 90% positive detection rates when diagnosing N. gonorrhoeae (9).

NAATs are based on targeting conserved and specific gene sequences found in the bacteria of interest (3). When there is a presence of the target nucleic acids (RNA or DNA), the primers with complementary base pairs will bind to the matching nucleic acids and induce amplification (10, 11). NAATs amplify specific nucleic acids by using a polymerase chain reaction, transcription-mediated assay or strand displacement assay (10, 11). NAATs typically detect the particular sequences within 16sRNA, opa genes, gyrA gene (encoding DNA gyrase subunit A), cppB gene (encoding cryptic plasmid protein B) and proA pseudogenes that are specific to N. gonorrhoeae (3, 6).

In the presence of other bacterial species with similar target genes, NAATs may generate false-positive results (3, 8). It is reported that there is a higher incidence of false-positive results among asymptomatic patients and there is a need to be cautious (11). Moreover, the U.S. Food and Drug Administration does not approve rectal, pharyngeal, or conjunctival specimens as a sample for NAATs (11). Thus, there is a limitation on the sample collection. Other disadvantages include contamination risk and the inability to detect antimicrobial susceptibility (8). The latter is an important step in determining which antibiotics the infectious agent is resistant to, which helps to establish an effective treatment plan for patients (8).

References:

1. Beveridge TJ, Davies JA. Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain. J Bacteriol. 1983 Nov;156(2):846-58. doi: 10.1128/jb.156.2.846-858.1983.

2. Meyer T, Buder S. The Laboratory Diagnosis of Neisseria gonorrhoeae: Current Testing and Future Demands. Pathogens. 2020 Jan 31;9(2):91. doi: 10.3390/pathogens9020091.

3. Unemo, Magnus et al. “Gonorrhoea.” Nature reviews. Disease primers vol. 5,1 79. 21 Nov. 2019, doi:10.1038/s41572-019-0128-6

4. Public Health Agency of Canada (PHAC). Gonorrhea guide: Screening and diagnostic testing. Ottawa, ON: Government of Canada; 2022 Apr; 2022. Available from: https://www.canada.ca/en/public-health/services/infectious-diseases/sexual-health-sexually-transmitted-infections/canadian-guidelines/gonorrhea/screening-diagnostic-testing.html

5. ghanem, Khalil G. “Clinical Manifestations and Diagnosis of Neisseria Gonorrhoeae Infection in Adults and Adolescents.” UpToDate, May 2022, https://www.uptodate.com/contents/clinical-manifestations-and-diagnosis-of-neisseria-gonorrhoeae-infection-in-adults-and-adolescents/print.

6. Centers for Disease Control and Prevention. “Recommendations for the laboratory-based detection of Chlamydia trachomatis and Neisseria gonorrhoeae--2014.” MMWR. Recommendations and reports : Morbidity and mortality weekly report. Recommendations and reports vol. 63,RR-02 (2014): 1-19.

7. Gonorrhea - ClinicalKey. 27 April 2022. https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/clinical_overview/67-s2.0-039c0229-bab2-40a5-a33b-99185aa6ec75#diagnostic-procedures-heading-19. Retrieved 10 February 2023.

8. Whiley DM, Tapsall JW, Sloots TP. Nucleic acid amplification testing for Neisseria gonorrhoeae: an ongoing challenge. J Mol Diagn. 2006 Feb;8(1):3-15. doi: 10.2353/jmoldx.2006.050045.

9. Papp JR, Schachter J, Gaydos CA, Van Der Pol B. 2014. Recommendations for the Laboratory-Based Detection of Chlamydia trachomatis and Neisseria gonorrhoeae — 2014. MMWR. Recomm. Rep. 63:1–19. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047970/

10. Bennet JE, Dolin R, Blaser MJ. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 9th Ed. Philadelphia, PA: Elsevier; 2020.

11. Duncan JA, Brown LB, Leone PA. 2011. Gonococcal and Other Neisserial Infections, p. 184–190. In Tropical Infectious Diseases: Principles, Pathogens and Practice. Elsevier.

(iv) What are the expected results from the above tests allowing for the identification of the bacteria named in this case? (For culture-based testing, consider microscopy, culture (colony morphology, biochemical testing, confirmatory testing)

Bacterial Culture

Cultures can identify N. gonorrhoeae through growth on media - the yield of the culture will depend on the type of media, which can be selective or non-selective, as well as the anatomical site used for specimen collection as well as the adequacy of the specimen itself (1, 2). Cultures produce positive diagnoses using urethral, endocervical, rectal, oropharyngeal, and conjunctival swabs (1). Urethral swabs are not recommended for female patients however (1). Identification is based on growth. When colonies appear on the selected culture medium, this would be considered a positive result (3, 4). Generally, 18-24 hours after incubation at 35–37 °C and high humidity (70–80%), pH 6.75–7.5, and in a 4–6% CO2-enriched atmosphere, small, shiny, grey colonies appear (5). Culture should also not be incubated for more than 48 hours as they would not survive storage conditions (6). To confirm the results, microscopic gram-staining preparation and positive cytochrome oxidase reaction can be possible (5). Since culture is generally not able to distinguish between various Neisseria strains, confirmatory testing needs to be performed after a culture is grown (5).

Confirmatory Testing - Biochemical Tests

Related to bacterial cultures, biochemical tests are used to identify and confirm the identity of the strains. Biochemical tests generally require detection of acid production from carbohydrates (glucose for N. gonorrhoeae) in a cystine trypticase acid (CTA) base medium (3, 4). Various enzymatic and metabolic reactions are shown through colour changes (5). The type of pathogen can be figured out based on a determined numerical panel (5). Other commercial biochemical tests can test for characteristics such as enzyme production, DNAse and nitrate reduction (6).

Alternatively, coagglutination tests that contain gonococcal-specific monoclonal antibodies can be performed as well (5). These tests are conducted with primary cultures, and coagglutination with test reagents indicate a positive identification (6).

Nucleic Acid Amplification Tests (NAATs)

NAATs can be used to amplify and detect gonococcal nucleic acid (DNA or RNA) sequences without the need for viable organisms. When the gene target is detected, the nucleic acid sequence from the organism is amplified and copies of the targeted sequence are produced, which produces a positive signal and result (6, 7). For NAATs, the presence of N. gonorrhoeae-specific gene including sequences within 16sRNA, opa genes, and proA pseudogene confirms that the infectious agent is N. gonorrhoeae (2, 3). Currently, multiplex PCR is commonly used, which allows concurrent identification of multiple species which is then delineated with gel electrophoresis (8). They also generally have a positive predictive value of >90% (3), meaning that N. gonorrhoeae can be positively and correctly identified >90% of the time through the use of NAATs.

Smears/Gram Stain + Microscopy

For smear microscopy, the specimen is first fixed on a slide by spreading out the sample on a microscopic slide and heated (5). The diagnostic specificity and sensitivity of the Gram stain are contingent upon the type of specimen used to this end (9).

Gram stains produce a positive result for N. gonorrhoeae when neutrophils with intracellular Gram-negative diplococci (pairs) are observed, although young cultures may produce organisms that occur in tetrads (1, 4). Diplococci occur perpendicular to each other, and generally have the same size and shape (5). Leukocytes may also be present in a positive result (1, 2, 10). Detection of extracellular diplococci, in addition to a typical clinical picture, can also be considered a positive result for N. gonorrhoeae (5). Negative results occur when no such diplococci or leukocytes are observed through microscopy (1, 2, 10). Equivocal results occur when gram-negative diplococci are present and leukocytes are absent, or otherwise morphologically atypical organisms are present (1, 2, 10). If the result is equivocal, then confirmatory testing such as culture or nucleic acid testing should be conducted to confirm the specimen’s identity (1, 2, 10).

Organisms with gram-negative cell walls like N. gonorrhoeae will become decolourized and stain red due to safranin counterstain (10S), so expected results for a positive diagnosis would be red-coloured colonies. However, it is difficult to distinguish between different Neisseria strains with Gram stains, and therefore culture is used as well to confirm the diagnosis (4).

Sources:

  1. Duncan JA, Brown LB, Leone PA. 2011. CHAPTER 25 - Gonococcal and Other Neisserial Infections, p. 184–190. In Guerrant RL et al. (ed), Tropical Infectious Diseases: Principles, Pathogens and Practice (Third Edition). W.B. Saunders, Edinburgh. https://doi.org/10.1016/B978-0-7020-3935-5.00025-2.
  2. Marrazzo JM, Apicella MA. 2020. Neisseria gonorrhoeae (Gonorrhea). In Bennett JE et al (ed), Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, Ninth Edition. Elsevier. https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/book/3-s2.0-B9780323482554002125?scrollTo=%23hl0001164.
  3. Unemo M, Seifert HS, Hook, EW III, Hawkes S, Ndowa F, Dillon J-A. 2019. Gonorrhoea. Nat. Rev. Dis. Primers 5(79). https://doi.org/10.1038/s41572-019-0128-6.
  4. Wojewoda CM, Stempak LM. Medical Bacteriology. In McPherson RA et al (ed), Henry's Clinical Diagnosis and Management by Laboratory Methods, Twenty Fourth Edition. Elsevier. https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/book/3-s2.0-B9780323673204000572?scrollTo=%23hl0001752.
  5. Meyer T, Buder S. The Laboratory Diagnosis of Neisseria gonorrhoeae: Current Testing and Future Demands. Pathogens. 2020 Jan 31;9(2):91. doi: 10.3390/pathogens9020091.
  6. Ng L-K, Martin IE. 2005. The laboratory diagnosis of Neisseria gonorrhoeae. Can. J. Infect. Dis. Med. Microbiol. 16:15–25. doi: 10.1155/2005/323082.
  7. Papp JR, Schachter J, Gaydos CA, Van Der Pol B. 2014. Recommendations for the Laboratory-Based Detection of Chlamydia trachomatis and Neisseria gonorrhoeae — 2014. MMWR. Recomm. Rep. 63:1–19. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047970/
  8. Leos-Alvarado C, Llaca-Díaz J, Flores-Aréchiga A, Pérez-Chávez F, Casillas-Vega N. Male urethritis. A review of the ideal diagnostic method. Actas Urológicas Españolas (English Edition). 2020 Oct 1;44(8):523-8.
  9. Public Health Agency of Canada (PHAC). Gonorrhea guide: Screening and diagnostic testing. Ottawa, ON: Government of Canada; 2022 Apr; 2022. Available from: https://www.canada.ca/en/public-health/services/infectious-diseases/sexual-health-sexually-transmitted-infections/canadian-guidelines/gonorrhea/screening-diagnostic-testing.html
  10. Morse SA. 1996. Neisseria, Moraxella, Kingella and Eikenella, p. . In Baron, S (ed.), Medical Microbiology, 4th ed. University of Texas Medical Branch at Galveston, Galveston (TX).


3. Bacterial Pathogenesis

(i) Encounter: where does the organism normally reside - geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How would our patient have come in contact with this bacteria?

New gonorrhea cases in 2016 (WHO Estimate).
Figure 1. New gonorrhea cases in 2016 (WHO Estimate).

Neisseria (N.) gonorrhoeae is an obligate human pathogen (1). Though it first came from animals, it has evolved to infect only human cells. The main adaptation that allows it to thrive is its ability to change antigenic phases of its structures, thereby largely evading immune defences (2). As of 2016, the WHO reported that gonorrhoea is a virtually global phenomenon with a slightly higher prevalence in men than women (0.9% vs 0.7%) (3). However, a larger proportion of cases were reported in the WHO African Region, the Americas, and Western Pacific, and lowest in the European region (Figure 1.) (3). Among these populations, selected groups- namely minority groups, sex workers, transgender, gay and bisexual persons- experienced higher incidences of the disease likely due to various factors affecting poor sexual health outcomes (3). Once infected, N. gonorrhoeae usually infects the mucous membrane of reproductive tracts in areas such as the cervix, urethra, uterus, and fallopian tubes. In some cases, it can infect mucous membranes in the mouth, throat, eyes, and rectum (4).The urogenital tract has many different types of epithelial tissues and immune cells throughout the mucosa. N. gonorrhoeae is a host-adapted organism and can interact with many molecules.(5) It can evade and modulate the innate and adaptive immune systems to better survive.(5) How the patient gets the bacteria: Patients get N. gonorrhoeae primarily through vaginal, anal, or oral sex, i.e. sexual fluids. Sometimes, it can be transmitted by touching one’s eye with infected fluids (6). Though the bacteria seems to be spread by contact, casual contact is not known to be a means of transmission. This includes sneezing, coughing, holding hands, etc. (6).

References:

  1. J. D. Lenz and J. P. Dillard, “Pathogenesis of neisseria gonorrhoeae and the host defense in ascending infections of human fallopian tube,” Frontiers, 02-Nov-2018. [Online]. Available: https://doi.org/10.3389/fimmu.2018.02710. [Accessed: 10-Feb-2023].
  2. Y. Liu, W. Liu, and M. W. Russell, “Suppression of host adaptive immune responses by Neisseria gonorrhoeae: Role of interleukin 10 and type 1 regulatory T cells,” Mucosal Immunology, vol. 7, no. 1, pp. 165–176, 2014.
  3. R. D. Kirkcaldy, E. Weston, A. C. Segurado, and G. Hughes, “Epidemiology of gonorrhoea: A global perspective,” Sexual Health, vol. 16, no. 5, p. 401, 2019.
  4. CDC, “Detailed std facts - gonorrhea,” Centers for Disease Control and Prevention, 01-Dec-2022. [Online]. Available: https://www.cdc.gov/std/gonorrhea/stdfact-gonorrhea-detailed.htm#:~:text=N.,throat%2C%20eyes%2C%20and%20rectum. [Accessed: 10-Feb-2023].
  5. Quillin, S., Seifert, H. Neisseria gonorrhoeae host adaptation and pathogenesis. Nat Rev

6. P. Parenthood, “What is gonorrhea & how do you get it?,” Planned Parenthood, n.d.. [Online]. Available: https://www.plannedparenthood.org/learn/stds-hiv-safer-sex/gonorrhea. [Accessed:

10-Feb-2023].

(ii) Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host?

Transmission of N. gonorrhoeae is usually through infected people who are asymptomatic or those who have symptoms of gonorrhea but ignore or discount them. Rates of transmission are higher during unprotected vaginal sex from penis to vagina than from the vagina to penis. Probability of transmission via oral (urethra to pharynx) and anal (urethra to rectum) sex are 63% and 84% respectively. (1, 3, 4) The higher probability of penile ejaculates to transmit the bacteria is because N. gonorrhoeae attach to the sperm cells using LOS to spread the infection to sexual partners. (2) Transmission from the vagina would require the surface of the bacteria to be free from sialic acid (desialylation), achieved through the action of the natural microbiota of the vagina, for successful entry and binding. Transmission during childbirth is possible because the newborn’s conjunctivae are exposed to the infected birth canal. (3, 5)

Figure 2. Neisseria gonorrhoeae infection overview showing key features

After transmission, N. gonorrhoeae cells attach to various mucosal epithelial surfaces. Attachment is mediated by Type IV pili, opacity-associated (Opa) proteins, LOS, and a major porin named PorB. (5) Type IV pili, found on the bacterial outer membrane, are hair-like appendages that can retract and lengthen and are the key structures for initial adherence, cellular invasion, microcolony formation, and immune evasion. Once attached to a host cell, Type IV pili retraction would bring N. gonorrhoeae closer to the cell surface. After initial contact through Type IV pili, Opa proteins would further promote adherence and immune evasion via antigenic and phase variations. PorB and LOS also affect colonization - PorB suppresses neutrophil activity and apoptosis while LOS affects immune cell recognition (Figure 2). (3, 5)5.

Overview of Neisseria gonorrhoeae pathogenesis factors
Figure 3. Overview of Neisseria gonorrhoeae pathogenesis factors

N. gonorrhoeae invasion of epithelial cells involves different receptors for males and females though both require LOS to be desialylated. In the male urethral epithelium, LOS interacts with the asialoglycoprotein receptor to promote colonization, while in females, the CR3 receptor (lower genital tract) and LHR receptor (endometrium and oviducts) mediate cell invasion.

References:

  1. Bennett JE, Dolin R, Blaser MJ. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 9th Ed. Philadelphia, PA: Elsevier; 2020.
  2. Springer C, Salen P. Gonorrhea [Internet]. Treasure Island, FL: StatPearls Publishing; 2022 Jan [updated 2022 Apr 21; cited 2023 Feb 8]. Available from https://www.ncbi.nlm.nih.gov/books/NBK558903/
  3. Unemo M, Seifert HS, Hook EW 3rd, Hawkes S, Ndowa F, Dillon JR. Gonorrhoea. Nat Rev Dis Primers [Internet]. 2019 Nov 21 [cited 2023 Feb 8];5(1):79. Available from: https://doi.org/10.1038/s41572-019-0128-6. PMID: 31754194.
  4. Haese EC, Thai VC, Kahler CM. Vaccine Candidates for the Control and Prevention of the Sexually Transmitted Disease Gonorrhea. Vaccines (Basel) [Internet]. 2021 Jul 20 [cited 2023 Feb 8];9(7):804. Available from: https://doi.org/10.3390/vaccines9070804.
  5. Quillin S, Seifert H. Neisseria gonorrhoeae host adaptation and pathogenesis. Nat Rev Microbiol. [Internet]. 2018 Feb 12 [cited 2023 Feb 8];16, 226–240. Available from: https://doi.org/10.1038/nrmicro.2017.169.


(iii) Multiplication and Spread: does the organism remain extracellular or do they enter into cells? What are the molecular and cellular determinants of these events? Do the bacteria remain at the entry site or do they spread beyond the initial site? (e.g. are there secondary sites of infection) and, if they spread, why do the bacteria hone in on particular secondary sites?

Neisseria gonorrhoeae is an interesting bacteria that can survive as an extracellular or intracellular organism (3). The survival of Neisseria gonorrhoeae is dependent on the surface components that are expressed and if the components are chemically modified (3). A frameshift in a gene can lead to the random switching between intracellular and extracellular states (3). Antigenic variation can change the chemical forms of the structural components (3). However, a more recent study has shown that Neisseria gonorrhoeae can only survive intracellularly, and not extracellularly (4). If it is not in contact with physiological temperatures, it will be unviable (4).

The bacteria can survive intracellularly by utilizing its virulence factors. As stated above, Neisseria gonorrhoeae can use Opa proteins, Por proteins, and LOS to facilitate the process of invasion into the cell (2). Once inside the cell, the bacteria can survive in the presence of macrophages and neutrophils (4). They can also manipulate dendritic cells and their activation properties (4). If taken up by a macrophage, Neisseria gonorrhoeae can survive in a phagosome and regulate the production of cytokines (4). As a result, macrophages will not be able to activate T cells and dendritic cells are unable to stimulate T cell proliferation (4).

Neisseria gonorrhoeae typically stays in mucous membranes of the reproductive system and in some cases, the mouth, throat, eyes, or rectum (1). However, if the bacteria persist, they can spread to other parts of the body through the blood stream (5). This occurs when N. gonorrhea invades epithelial cells and is transcytosed to the other side of the cell to where it can then cross the barrier into the bloodstream (2,6,7). This can cause gonococcal arthritis (8). It can either result in localized septic arthritis or arthritis-dermatitis syndrome(8). Arthritis-dermatitis syndrome is composed of tenosynovitis, dermatitis, and polyarthralgia. Side effects are fever and chills (8). Localized septic arthritis is when there is pain and swelling in one or more joints, such as the knees and ankles (8). There are no side effects of fever or chills (8).

References:

  1. 2022. Detailed std facts - gonorrhea. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention.
  2. Quillin SJ, Seifert HS. 2018. Neisseria gonorrhoeae host adaptation and pathogenesis. Nature Reviews Microbiology 16:226–240
  3. Hill SA, Masters TL, Wachter J. 2016. Gonorrhea – an evolving disease of the New Millennium. Microbial Cell 3:371–389.
  4. Quillin SJ, Seifert HS. 2018. Neisseria gonorrhoeae host adaptation and pathogenesis. Nature Reviews Microbiology 16:226–240.
  5.  2021. Gonorrhea. Mayo Clinic. Mayo Foundation for Medical Education and Research.
  6. Ruch TR, Engel JN. Targeting the Mucosal Barrier: How Pathogens Modulate the Cellular Polarity Network. Cold Spring Harb Perspect Biol. 2017 Jun;9(6):a027953.
  7. Walker E, van Niekerk S, Hanning K, Kelton W, Hicks J. Mechanisms of host manipulation by Neisseria gonorrhoeae. Frontiers in Microbiology [Internet]. 2023 [cited 2023 Feb 17];14. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1119834
  8. Gonococcal arthritis - statpearls - NCBI bookshelf.


(iv) Bacterial Damage: do the bacteria cause any direct damage to the host (vs. the damage fully attributable to the host response); if not, what are the main bacterial mechanisms that facilitate host-mediated damage?

Neisseria gonorrhoeae (N. gonorrhoeae) mainly causes damage that is attributable to the host response. Although N. gonorrhoeae does not express potent exotoxins that damage the host, it can lead to complications if left untreated that can lead to female genital tract infections, pelvic inflammatory disease, and even infertility (3). N. gonorrhoeae is able to stimulate a large innate immune response from the host which can supress the adaptive immune system, which can further lead to additional infections and also chronic repeated infections (4).

Some of the specific mechanisms used by this bacteria are caused by gonococcal lipooligosaccharides (LOS). LOS are able to promote invasion at higher levels than other strains that lack this structure as LOS plays an important role in initiating the invasion in humans. It affects cells by affecting its immune cell recognition which provides the bacteria resistance through complement evasion which can lead to further host transmission. However, the exact mechanisms of LOS remains to be further examined (1)

Another way that N. gonorrhoeae causes damage is through inflammation. Toll-like-receptor (TLR) and nucleotide-binding oligomerization domain-like receptor (NOD) are signaled in epithelial cells, macrophages, and dendritic cells. This could elicit a host immune response which leads to inflammatory transcription factors, cytokines, and chemokines to be released which would lead to inflammation to the tissue cells. The immune system further recruits a large number of neutrophils to the site of infection, however; due to the large influx of cells, it can compromise a purulent exudate that could lead to further transmission (3). Additionally N. gonorrhoeae is able to bind to and inactivate complement cascade components and prevent membrane attack complexes from forming, which could be detrimental against protection from other foreign bodies (2).

References:

  1.  Hsu, K., Ram, S., & Darville, T. (n.d.). Neisseria gonorrhoeae (Gonococcus). Clinicalkey . Retrieved February 10, 2023, from https://www.clinicalkey.com/student/content/book/3-s2.0-
  2. Quillin, S. J., & Seifert, H. S. (2018, February 12). Neisseria gonorrhoeae host adaptation and pathogenesis. Nature News. Retrieved February 9, 2023, from https://www.nature.com/articles/nrmicro.2017.169#:~:text=larger%2 0sexual%20network.-,N.,The%20surface%20of%20N.
  3. Yang, T., Heydarian, M., Kozjak-Pavlovic, V., Urban, M., Harbottle, R. P., & Rudel, T. (2020, July 8). Folliculin controls the intracellular survival and trans-epithelial passage of Neisseria Gonorrhoeae. Frontiers. Retrieved February 9, 2023, from https://www.frontiersin.org/articles/10.3389/fcimb.2020.00422/full#:~:text=Although%20N.,et%2 0al.%2C%202008).
  4. Gunderson, C. W., & Seifert, H. S. (2015, February 10). Neisseria gonorrhoeae elicits extracellular traps in primary neutrophil culture while suppressing the oxidative burst. mBio. Retrieved February 9, 2023, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337578/



4. The Immune Response

(i) Host response: what elements of the a.) innate and b.) adaptive (humoral and cellular) immune response are involved in this infection? Describe the immune cell populations involved as well as key chemical mediators.

Figure A. PMNs responses during health and disease. Pathological factors inducing inflammatory PMN response during chronic infection [7]

Gonorrhea, an infection caused by the Neisseria gonorrhoeae bacterium (N. gonorrhoeae), is a sexually transmitted disease that can be acquired from sexual contact with the genitalia, mouth, or anus of an infected partner [1]. Infection can also be spread from mother to baby during childbirth [1].

Studies of the human immune response to N. gonorrhoeae depict an organism that is adapted to thrive in human hosts [2]. Cervical and pharyngeal infections of gonorrhea are asymptomatic, therefore evaluating host responses can be difficult [3]. However, studies focused on human immune responses to gonococcal infection reveal that elements of both the innate and adaptive immune responses are involved.

Figure B. Induction of proinflammatory cytokines and chemokines in mouse sample [8].

The innate immune response is the first line of defense against bacterial infection and is initiated by the recognition of bacterial factors [4]. These bacterial factors include lipooligosaccharide (LOS) and peptidoglycan which are recognized by extracellular or intracellular pattern recognition receptors (PRRs) including toll-like receptors (TLR) TLR2 and TLR4, and nucleotide-binding oligomerization domain-containing proteins (NOD) NOD1 and NOD2 [4]. This system is not as specific as the adaptive immune response and is able to be initiated more quickly following bacterial introduction to the host. N. gonorrhoeae initially infects mucosal epithelium, where it is detected by sentinel immune cells including T helper 17 (TH17) cells, macrophages, and dendritic cells [5]. When an infection occurs, the innate immune system directs immune cells to move to the area of infection and activates these cells, including immune cells that were already near the infected area [1]. This induces the innate immune system to release pro-inflammatory cytokines and chemokines (IL-6, IL-8, IL-1B, IL-17, interferon-γ, and NF-κβ) which stimulate the invasion of polymorphonuclear neutrophils (PMNs) to the infected

Figure C. N. gonorrhoeae infection overview [4].
Table A. Presence of anti-gonococcal immunoglobulins and their associated antigen triggers [10]

endothelial of the reproductive tract [6]. Neutrophils act to combat bacterial infection through extracellular killing mechanisms and by engulfing bacteria that are then sequestered into phagosomes containing reactive oxygen species (ROS), degrading enzymes, and antimicrobial peptides [5]. The pathogenic process of a hyper PMN response dictates chronic inflammation that induces immune-mediated tissue damage and microbial dysbiosis, increasing inflammation [3, 7, figure A]. N. gonorrhoeae also secretes heptose-1,7-bisphosphate (HBP), a pathogen-associated molecular pattern that activates Traf-interacting protein with forkhead-associated domain (TIFA)-dependent immunity, an innate immune mechanism in macrophages and epithelial cells [4]. Furthermore, N. gonorrhoeae induces interleukin-1α (IL-1α) and tumor necrosis factor-alpha (TNF-α), therefore initiating a signaling cascade [8]. The signaling activates and recruits phagocytic cells, PMNs, macrophages, peripheral blood mononuclear cells, and an array of pro-inflammatory cytokines (IL-1α, TNF-α, and IL-6) and chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein 2 (MIP-2)) [8]. Notably, increased localization of MIP-2 is correlated to PMN influx [8, figure B]. The infection also activates the alternative and classic complement pathways – key components of the innate immune system. The complement pathways recognize and kill pathogens through opsonization and the formation of membrane attack complexes [4]. Those with known complement deficiency are more susceptible to disseminated gonococcal infection [9].

Table B. Presence of anti-gonococcal cellular immune responses and their associated antigen triggers [10].

Humoral adaptive immune responses include the production of anti-gonococcal immunoglobulins [10]. These antibody responses produce IgA, IgG and IgM [10] and indicate the involvement of B cells and plasma cells. IgG, IgA, and IgM antibody levels are elevated in cases of urethritis and cervicitis [10]. Antibodies are primarily produced against the antigens LOS and bacterial pili [10, table A], but may also be produced against bacterial porins and other structures [10, 11]. However, these antibody responses are weak in early infection, only becoming strong at later stages of infection when inflammatory damage has already occurred [4]. It has been shown that infected hosts produce serum complement-dependent bactericidal antibodies, though they have not been shown to prevent the recurrence of the disease [12]. N. gonorrhoeae largely suppresses T helper 1 and 2 immune responses [3, 4, 12], while selecting for T helper 17 cell populations [3, 12], by increasing levels of transforming growth factor-beta (TGF-β) and IL-10 [12]. The role of CD8 T cells and natural killer (NK) cells in N. gonorrhoeae infection is unknown [10]. The cell-mediated adaptive immune response includes lymphocyte proliferation and stimulated cytokine release of IL-4, IL-10, IFN-γ, and TNF-α by PMBC, CD4, and CD8 T cells [10, table B].

References

  1. Klein EJ, Fisher LS, Chow AW, Guze LB. Anorectal gonococcal infection. Ann Intern Med, 86, 340–346 (1977).
  2. Bennett JE, Dolin R, Blaser MJ. 2020. Chapter 212, Neisseria gonorrhoeae (Gonorrhea). In Mandell, Douglas, and Bennett's principles and practice of infectious diseases. Elsevier, Philadelphia, PA. Retrieved on February 2, 2023, from https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323482554002125
  3. Russell MW, Gray-Owen SD, Jerse AE. 2020. Editorial: Immunity to neisseria gonorrhoeae. Frontiers in Immunology 11. https://doi/10.3389/fimmu.2020.01375
  4. Quillin SJ, Seifert HS. 2018. Neisseria gonorrhoeae host adaptation and pathogenesis. Nature Reviews Microbiology 16:226–240. https://doi.org/10.1038/nrmicro.2017.169
  5. Criss AK, Seifert HS. A bacterial siren song: intimate interactions between Neisseria and neutrophils. Nat Rev Microbiol. 2012;10(3):178-190. Published 2012 Jan 31. doi:10.1038/nrmicro2713
  6. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell. 2010 Mar 19;140(6):805-20. doi: 10.1016/j.cell.2010.01.022. PMID: 20303872.
  7. Fine N, Tasevski N, McCulloch CA, Tenenbaum HC, Glogauer M. 2020. The neutrophil: Constant defender and first responder. Frontiers in Immunology 11. https://doi.org/10.3389/fimmu.2020.571085
  8. Packiam M, Veit SJ, Anderson DJ, Ingalls RR, Jerse AE. 2010. Mouse strain-dependent differences in susceptibility to neisseria gonorrhoeae infection and induction of innate immune responses. Infection and Immunity 78:433–440. https://doi.org/10.1128/IAI.00711-09
  9. Lewis LA, Ram S. Complement interactions with the pathogenic Neisseriae: clinical features, deficiency states, and evasion mechanisms. FEBS Lett. 2020;594(16):2670-2694. doi:10.1002/1873-3468.13760
  10. Lovett A, Duncan JA. 2019. Human immune responses and the natural history of neisseria gonorrhoeae infection. Frontiers in Immunology 9. https://doi.org/10.3389/fimmu.2018.03187
  11. Marrazzo JM, Apicella MA. Neisseria gonorrhoeae (Gonorrhea). In: Bennett JE, Dolin R, Blaser MJ, editors. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 9th ed. Philadelphia (PA): Elsevier; 2020. p.2608-2627.e3.
  12. Russell MW. Immune Responses to Neisseria gonorrhoeae: Challenges and Opportunities With Respect to Pelvic Inflammatory Disease. J Infect Dis. 2021;224(12 Suppl 2):S96-S102. doi:10.1093/infdis/jiaa766

(ii) During a bacterial infection, excessive inflammation can result in immune-mediated pathology. What (if any) immune-mediated damage is caused by N. gonorrhoeae infection? Describe what cell types are involved, and whether pathology is occurring locally or systemically. Consider less severe and more severe cases.

N. gonorrhoeae does not express any potent exotoxins itself [1]; rather, the pathology of N. gonorrhoeae infection is largely mediated by the innate immune response to infection at sites of colonization [2]. Gonococcus can persist for months leading to a continual inflammatory response [3]. The excessive inflammation is primarily caused by the robust, extensive PMN response [3, 4]. This characteristic inflammatory response damages host tissue and can lead to complications or dissemination of the infection [5]. Neutrophils and macrophages phagocytose the pathogen, however extensive and prolonged infection further promotes the innate immune response. Therefore, left untreated the PMN attacks the mucosal and epithelial levels of the infection site. Neutrophils secrete reactive oxygen species (ROS) to increase inflammation and neutrophil recruitment, but these ROS also alter lipids, proteins, and DNA [6]. Neutrophil degranulation also secretes matrix metalloproteinases that degrade the extracellular matrix [6]. In their neutrophil extracellular traps (NETs), neutrophils secrete histones which cause direct epithelial cell damage [6]. This leads to neutrophil-mediated tissue damage and inflammation. Early in infection, this often presents as urethritis [7], but if the infection progresses to higher reproductive organs, this inflammation becomes more pronounced [6].  In most cases the pathology of immune-mediated damage is primarily localized to the reproductive tract; however, l​ocal oropharyngeal infection can also result from oral sexual activities [8]. In mild cases, infection may result in mild tissue damage, inflammation and redness [9]. In these cases, immune cells such as neutrophils and monocytes, which are attracted to the site of infection by chemokines, are involved in the immune response [7]. Recruited neutrophils do not effectively eliminate the infection, rather they present a host for pathogen replication and may facilitate more efficient transmission through passing of infected neutrophils to a sexual partner [2].

If a localized case of N. gonorrhoeae becomes more severe it can lead to complications such as pelvic inflammatory disease, scarring of the epididymis or fallopian tubes, infertility, and ectopic pregnancy [1, 10]. This is because infection and inflammation of the fallopian tube can cause scarring, occlusion, and loss of critical ciliated cells [11].  It may also cause cervicitis [12]. Further, untreated infections can be passed during birth to an infant where the bacteria may colonize and trigger inflammation of the eyes, leading to blindness [2].

In some severe cases of N. gonorrhoeae infection, excessive inflammation can lead to systemic pathology. For example, disseminated gonococcal infection (DGI), which is a severe form of N. gonorrhoeae infection that affects multiple organs and tissues, is associated with a systemic immune response [13, 14]. Dissemination of the infection is thought to occur partly through N. gonorrhoeae interactions with neutrophils [5]. The bacteria can live and replicate within the neutrophil, effectively evading the host immune system, which can allow for deeper penetration of tissues and possibly aids in transmission [5]. In DGI, the bacterium can spread from the initial site of infection to other parts of the body through the bloodstream which can result in inflammation and damage to tissues in multiple organs, such as the skin, joints, and heart [14]. The immune response to N. gonorrhoeae in DGI is characterized by the activation of immune cells, such as T cells and macrophages, and the release of pro-inflammatory cytokines, such as TNF-α, IL-1, and IL-8 [15]. If gonococcal bacteremia is present, there is most likely an issue with host complement activity as complement activity is known to combat infection of N. gonorrhoeae [16]. In rare cases, infectious arthritis and endocarditis may occur [10]. Furthermore, N. gonorrhoeae can be transmitted from mother to infant during delivery [17]. Potentially, leading to neonatal blindness [10]. Furthermore, an N. gonorrhoeae infection activates CD4+ T cells initiating an NF-κβ response that drives HIV-1 expression, therefore increasing the risk of AIDS [18].

References:

  1. Russell MW, Gray-Owen SD, Jerse AE. 2020. Editorial: Immunity to neisseria gonorrhoeae. Frontiers in Immunology 11. https://doi/10.3389/fimmu.2020.01375
  2. Quillin SJ, Seifert HS. Neisseria gonorrhoeae host adaptation and pathogenesis. Nat Rev Microbiol [Internet]. 2018 Feb;16(4):226-240. doi:10.1038/nrmicro.2017.169
  3. Bennett JE, Dolin R, Blaser MJ. 2020. Chapter 212, Neisseria gonorrhoeae (Gonorrhea). In Mandell, Douglas, and Bennett's principles and practice of infectious diseases. Elsevier, Philadelphia, PA. Retrieved on February 2, 2023, from https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323482554002125
  4. Fine N, Tasevski N, McCulloch CA, Tenenbaum HC, Glogauer M. 2020. The neutrophil: Constant defender and first responder. Frontiers in Immunology 11. https://doi.org/10.3389/fimmu.2020.571085
  5. Criss AK, Seifert HS. A bacterial siren song: intimate interactions between Neisseria and neutrophils. Nat Rev Microbiol. 2012;10(3):178-190. Published 2012 Jan 31. doi:10.1038/nrmicro2713
  6. Stevens JS, Criss AK. Pathogenesis of Neisseria gonorrhoeae in the female reproductive tract: neutrophilic host response, sustained infection, and clinical sequelae. Curr Opin Hematol [Internet]. 2018 Jan;25(1):13-21. doi:10.1097/MOH.0000000000000394
  7. Lovett A, Duncan JA. Human Immune Responses and the Natural History of Neisseria gonorrhoeae Infection. Front Immunol [Internet]. 2019 Feb;9:3187. doi:10.3389/fimmu.2018.03187
  8. Hook EW 3rd, Bernstein K. Kissing, saliva exchange, and transmission of Neisseria gonorrhoeae. Lancet Infect Dis [Internet]. 2019 Jul;19(10):e367-e369. doi:10.1016/S1473-3099(19)30306-8
  9. Zell A. McGee, David S. Stephens, Loren H. Hoffman, Walter F. Schlech, III, Robert G. Horn, Mechanisms of Mucosal Invasion by Pathogenic Neisseria, Reviews of Infectious Diseases, Volume 5, Issue Supplement_4, September-October 1983, Pages S708–S714, https://doi.org/10.1093/clinids/5.Supplement_4.S708
  10. Schenkel K, Gupta R. 2021. Molecular probes and detection of sexually transmitted infections. Fluorescent Probes 111–133. https://doi.org/10.1016/bs.mim.2021.03.002
  11. Lenz JD, Dillard JP. Pathogenesis of Neisseria gonorrhoeae and the Host Defense in Ascending Infections of Human Fallopian Tube. Front Immunol [Internet]. 2018 Nov;9:2710. Published 2018 Nov 21. doi:10.3389/fimmu.2018.02710
  12. Russell MW. Immune Responses to Neisseria gonorrhoeae: Challenges and Opportunities With Respect to Pelvic Inflammatory Disease. J Infect Dis [Internet]. 2021 Aug;224(12 Suppl 2):S96-S102. doi:10.1093/infdis/jiaa766
  13. Birrell JM, Gunathilake M, Singleton S, Williams S, Krause V. Characteristics and Impact of Disseminated Gonococcal Infection in the "Top End" of Australia. Am J Trop Med Hyg. 2019 Oct;101(4):753-760. doi: 10.4269/ajtmh.19-0288. PMID: 31392956; PMCID: PMC6779203.
  14. Peter A. Rice MD. Gonococcal arthritis (disseminated gonococcal infection). Infectious Disease Clinics of North America. https://www.sciencedirect.com/science/article/abs/pii/S0891552005000681?via%3Dihub. Published November 16, 2005. Accessed February 10, 2023.
  15. Eisenstein BE, Masi AT. Disseminated gonococcal infection (DGI) and gonococcal arthritis (GCA): I. Bacteriology, epidemiology, host factors, pathogen factors, and pathology. Seminars in Arthritis and Rheumatism. https://www.sciencedirect.com/science/article/pii/S0049017281800017. Published August 22, 2006. Accessed February 10, 2023.  
  16. The Neisseriae. In: Riedel S, Hobden JA, Miller S, Morse SA, Mietzner TA, Detrick B, Mitchell TG, Sakanari JA, Hotez P, Mejia R. eds. Jawetz, Melnick, & Adelberg's Medical Microbiology, 28e. McGraw Hill; 2019. Accessed February 10, 2023. https://accessmedicine-mhmedical-com.ezproxy.library.ubc.ca/content.aspx?bookid=2629&sectionid=217771561
  17. Centers for Disease Control and Prevention. 2022. Detailed std facts - stds & pregnancy. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention. Retrieved from February 8, 2023 from Detailed STD Facts - STDs & Pregnancy (cdc.gov)
  18. Malott RJ, Keller BO, Gaudet RG, McCaw SE, Lai CC, Dobson-Belaire WN, Hobbs JL, St. Michael F, Cox AD, Moraes TF, Gray-Owen SD. 2013. neisseria gonorrhoeae- derived heptose elicits an innate immune response and drives HIV-1 expression. Proceedings of the National Academy of Sciences 110:10234–10239. https://doi.org/10.1073/pnas.1303738110

(iii) Bacterial evasion: how does the bacteria attempt to evade the host immune response? Consider mechanisms targeting innate and adaptive immune responses.

To evade the innate immune response’s complement system, N. gonorrhoeae utilizes three mechanisms to bind to complement proteins, therefore preventing opsonization and killing by membrane attack complexes [1, 2]. Sialylated LOS hinders the classical, leptin, and alternative complement pathways [3]. In the first mechanism, gonococcal LOS binds to complement protein C3b through lipid A; this inactivates C3b by factor I-mediated cleavage to produce iC3b [1, figure D] to hinder the complement cascade. The second mechanism sabotages complement activation in the cervical epithelium and human serum [1]. In the cervical epithelium, gonococcal LOS and porin bind to host factor H [1, figure D]. By binding to factor H, N. gonorrhoeae is recognized as self and is not targeted for opsonization and lysis by host structures [1]. In serum, N. gonorrhoeae can bind to host regulatory factors C4BP and CD46 to avoid recognition by host structures and therefore evade the immune system [1]. Finally, N. gonorrhoeae has specialized adhesins, Opa and Pil, that allow it to adhere to host cells and evade phagocytosis by immune cells, such as neutrophils and macrophages [4, 5]. This increases the survival of the bacterium within the host.

N. gonorrhoeae can also evade macrophages, dendritic cells, and neutrophils. When incubated with neutrophils in vitro, over 80% of N. gonorrhoeae bacteria persist [6]. N.

Figure D. Mechanisms of evasion and modulation of the innate and immune systems by N. gonorrhoeae. A) Inhibition of complement activation. B) Modulation of Macrophages, DCs, and Neutrophils. C) Modulation of T cell function to evade adaptive immune response [1].

gonorrhoeae possess metalloproteinase NGO1686 which allows for the bacteria to survive despite neutrophil extracellular killing mechanisms [6]. Even if N. gonorrhoeae are engulfed by neutrophils, some can persist and multiply within phagosomes, perhaps due to resistance to antimicrobial factors meant to kill the bacteria and an ability to prevent neutrophil apoptosis [6]. N. gonorrhoeae are also able to resist ROS killing by neutrophils, even if they lack antioxidant gene products [6]. N. gonorrhoeae delay fusion with primary granules in the neutrophil [7] which prevents release of the toxic antimicrobial molecules contained within these granules. N. gonorrhoeae also express lytic enzymes, usually used for peptidoglycan biosynthesis, that also enable bacterial resistance to lysozyme- and neutrophil-mediated killing by innate immunity [7]. In macrophages, N. gonorrhoeae can survive within the phagosome, allowing it to modulate apoptosis and the production of inflammatory cytokines, IL-10, PDL1, and TGFβ [1, figure D]. The pathogen polarizes the macrophage and hinders T-cell activation. Macrophages are essential for T cell activation as antigen-presenting cells [8]. The concept also applies to dendritic cells and therefore inhibits T cell proliferation [1]. Notably, more research is required to understand how N. gonorrhoeae modulates neutrophil activity. Theories suggest that the uptake and survival of the bacterium into the phagosome allows for the modulation of apoptosis, oxidative killing, or non-oxidative killing, leading to NETosis [1, 2]. However, more research is required to understand the mechanisms and their components.

N. gonorrhoeae also uses molecular mimicry to avoid the human immune response. N. gonorrhoeae surface glycolipids closely resemble human antigens [7]; this significantly hinders the ability of the innate immune system to identify these bacteria as foreign. Further, as a species, N. gonorrhoeae utilizes horizontal gene exchange mechanisms to assist in immune evasion [7]; this horizontal gene passing enables passing of genes encoding resistance to specific antibiotics or toxins from one bacteria to another [9].

Additionally, N. gonorrhoeae encodes an efflux pump for transporting any antimicrobial peptides, produced by the innate immune system, out of the cell [1]. This provides resistance against host immune responses.  

N. gonorrhoeae utilize phase and antigenic variation to evade the immune system and prevent formation of immunological memory or a robust adaptive immune response against the bacterium. Type IV pili, opacity-associated adhesion (OPA) proteins, and LOS, are surface structures that are capable of undergoing phase variation to avoid the adaptive immune system [1, 7]. Phase variation allows for generation of multiple phenotypes within a single clonal lineage, providing different advantages to different infection subpopulations and enabling long-term adaptation [8].  The N. gonorrhoeae chromosome contains one expression locus (pilE) which, through the process of Pilin Av (high-frequency gene conversion reactions), can constantly change expression to avoid detection [10]. This mechanism of antigenic variation is thought to be a major factor in the persistence of N. gonorrhoeae infections [10].  Additionally, N. gonorrhoeae adds phosphoethanolamine to its lipid A surface antigen [1]; this is an example of antigenic variation; the new antigen will not have been previously recognized by immune cells, thus there would be no adaptive immune response against this novel antigen.

Further, to evade the adaptive immune response, N. gonorrhoeae modulates T cell function [1]. N. gonorrhoeae can suppress T helper 1 and T helper 2 cell proliferation and activation [1, figure D]. Suppression occurs by modulating IL-10 and transforming growth factor-β cytokine, which influences the regulatory factors of CD4+ T cells [1]. Further, Opa proteins on N. gonorrhoeae bind and inactivate T helper cells [11]. A study done in a mouse model showed faster clearance of the infection and induced protection upon treatment of the mouse with IL 10 antibody, demonstrating that IL-10 is a key factor in the immune response against this pathogen [12]. N. gonorrhoeae is also thought to express IgA1 protease that can inactivate IgA, reduce CD4+ T cell function, and destroy B cells [6]. Finally, dendritic cells exposed to gonococcal infection lose the ability to induce CD4+ T cell proliferation [1].

References

  1. Quillin SJ, Seifert HS. 2018. Neisseria gonorrhoeae host adaptation and pathogenesis. Nature Reviews Microbiology 16:226–240. https://doi.org/10.1038/nrmicro.2017.169
  2. Płaczkiewicz J. 2019. Avoidance of mechanisms of innate immune response by neisseria gonorrhoeae. Postępy Mikrobiologii - Advancements of Microbiology 58:367–373. https://doi.org/10.21307/PM-2019.58.4.367
  3. Ram S, Shaughnessy J, de Oliveira RB, Lewis LA, Gulati S, Rice PA. Gonococcal lipooligosaccharide sialylation: virulence factor and target for novel immunotherapeutics. Pathog Dis [Internet]. 2017 Jun;75(4):ftx049. doi:10.1093/femspd/ftx049
  4. Alcott AM, Werner LM, Baiocco CM, Belcher Dufrisne M, Columbus L, Criss AK. Variable Expression of Opa Proteins by Neisseria gonorrhoeae Influences Bacterial Association and Phagocytic Killing by Human Neutrophils. J Bacteriol. 2022;204(4):e0003522. doi:10.1128/jb.00035-22
  5. Song W, Ma L, Chen R, Stein DC. Role of lipooligosaccharide in Opa-independent invasion of Neisseria gonorrhoeae into human epithelial cells. J Exp Med. 2000;191(6):949-960. doi:10.1084/jem.191.6.949
  6. Criss AK, Seifert HS. A bacterial siren song: intimate interactions between Neisseria and neutrophils. Nat Rev Microbiol. 2012;10(3):178-190. Published 2012 Jan 31. doi:10.1038/nrmicro2713
  7. Marrazzo JM, Apicella MA. Neisseria gonorrhoeae (Gonorrhea). In: Bennett JE, Dolin R, Blaser MJ, editors. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 9th ed. Philadelphia (PA): Elsevier; 2020. p.2608-2627.e3.
  8. Guerriero JL. 2019. Macrophages. International Review of Cell and Molecular Biology 73–93. https://doi.org/10.1016/bc.ircmb.2018.07.001
  9. Cehovin A, Lewis SB. Mobile genetic elements in Neisseria gonorrhoeae: movement for change. Pathog Dis [Internet]. 2017Aug;75(6):ftx071. doi:10.1093/femspd/ftx071
  10. Rotman E, Webber DM, Seifert HS. Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology. J Bacteriol. 2016;198(18):2470-2482. Published 2016 Aug 25. doi:10.1128/JB.00330-16
  11. Russell MW. Immune Responses to Neisseria gonorrhoeae: Challenges and Opportunities With Respect to Pelvic Inflammatory Disease. J Infect Dis [Internet]. 2021 Aug;224(12 Suppl 2):S96-S102. doi:10.1093/infdis/jiaa766
  12. Liu Y, Liu W, Russell MW. Suppression of host adaptive immune responses by Neisseria gonorrhoeae: role of interleukin 10 and type 1 regulatory T cells. Mucosal Immunology. January 2014:165-176.

(iv) Outcome: a.) is the bacteria completely removed, or can chronic infection occur? b.) does the patient recover fully? c.) can asymptomatic infection occur? and d.) is there sterilizing immunity to future infections from this particular bacteria?

N. gonorrhoeae infection can lead to bacterial clearance or prolonged infection. The immune system is quite poor at clearing N. gonorrhoeae infection by itself, thus antibiotics are often required for complete clearance of infection [1]. If untreated, it is possible for N. gonorrhoeae infection to persist for many months [1]. If the infection is left untreated for too long, it may spread to the reproductive tract causing pelvic inflammatory disease, ectopic pregnancies, or infertility as a result of inflammation and scarring [2, 3]. Additionally, if left untreated for long periods of time N. gonorrhoeae infection can become systemic dissemination and lead to disseminated gonococcal infection (DGI) which may manifest in septic arthritis [2, 4], endocarditis [2], skin manifestations [4], and more. Thus, patients with an acute infection that is treated promptly may recover fully, but chronic infection may produce irreversible effects.

Further, recurrent infection by the bacteria is a common occurrence, likely owing to the high transmissibility of the infection through sexual contact [5, 6]. Asymptomatic

Exposure and pathological timeline of N. gonorrhoeae exposure [4].

infection is estimated to occur in 20% of men and 50% of women in infected populations [4]. Asymptomatic infections can persist for over 165 days, while symptomatic infections can persist for two weeks to a year [4]. Asymptomatic infection often occurs in women, and if there are symptoms, they are usually non-specific [7]. Infection of the urethra often presents as dysuria and purulent discharge, while asymptomatic infection commonly occurs in the cervix, rectum, and pharynx [8]. Note, 80% of men and 50% of women in infected populations experience a symptomatic infection [4]. However, indicative of the poor immune response and reinfection by N. gonorrhoeae sterilizing immunity is unlikely. Reinfection with N. gonorrhoeae after successful antibiotic treatment is also common [1]. While humoral immune responses are produced, N. gonorrhoeae can suppress formation of immunological memory [2]. Therefore, decreasing the chances of sterilizing immunity for future infections and permitting re-infection if exposed. While IgA antibodies have been shown to be expressed post-infection, it is theorized that they are unable to protect against reinfection and are highly specified to gonococcal antigens [6]. This issue increases the difficulty of vaccine development. Only in 2019 did a study indicate a vaccine that reduced rates of gonorrhea in the population with a 24% reduced rate of hospitalizations due to infection [9]. Therefore, indicating a potential for vaccine development in the future.

References

  1. Marrazzo JM, Apicella MA. Neisseria gonorrhoeae (Gonorrhea). In: Bennett JE, Dolin R, Blaser MJ, editors. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 9th ed. Philadelphia (PA): Elsevier; 2020. P.2608-2627.e3.
  2. Quillin SJ, Seifert HS. 2018. Neisseria gonorrhoeae host adaptation and pathogenesis. Nature Reviews Microbiology 16:226–240. https://doi.org/10.1038/nrmicro.2017.169
  3. Cehovin A, Lewis SB. Mobile genetic elements in Neisseria gonorrhoeae: movement for change. Pathog Dis [Internet]. 2017Aug;75(6):ftx071. doi:10.1093/femspd/ftx071
  4. Lovett A, Duncan JA. 2019. Human immune responses and the natural history of neisseria gonorrhoeae infection. Frontiers in Immunology 9. https://doi.org/10.3389/fimmu.2018.03187
  5. Bennett JE, Dolin R, Blaser MJ. 2020. Chapter 212, Neisseria gonorrhoeae (Gonorrhea). In Mandell, Douglas, and Bennett's principles and practice of infectious diseases. Elsevier, Philadelphia, PA. Retrieved on February 2, 2023, from https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323482554002125
  6. The Neisseriae. In: Riedel S, Hobden JA, Miller S, Morse SA, Mietzner TA, Detrick B, Mitchell TG, Sakanari JA, Hotez P, Mejia R. eds. Jawetz, Melnick, & Adelberg's Medical Microbiology, 28e. McGraw Hill; 2019. Accessed February 10, 2023. https://accessmedicine-mhmedical-com.ezproxy.library.ubc.ca/content.aspx?bookid=2629&sectionid=217771561
  7. Unemo M, Seifert HS, Hook EW 3rd, Hawkes S, Ndowa F, Dillon JR. Gonorrhoea. Nat Rev Dis Primers. 2019;5(1):79. Published 2019 Nov 21. doi:10.1038/s41572-019-0128-6
  8. Criss AK, Seifert HS. A bacterial siren song: intimate interactions between Neisseria and neutrophils. Nat Rev Microbiol. 2012;10(3):178-190. Published 2012 Jan 31. doi:10.1038/nrmicro2713
  9. Paynter J, Goodyear-Smith F, Morgan J, Saxton P, Black S, Petousis-Harris H. 2019. Effectiveness of a group B outer membrane vesicle meningococcal vaccine in preventing hospitalization from gonorrhea in New Zealand: A retrospective cohort study. Vaccines 7:5. https://doig.org/10.3390/vaccines7010005
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