Sexually Transmitted Infection (STI) Case Study

Case study 5

A male, 24 years of age presents to the STI clinic. He complains of a burning and sore sensation upon urination, along with discharge from his penis that has a mucopurulent consistency and is green-yellow in colour. In the preceding two weeks, he has had unprotected sex with numerous partners.

Laboratory tests

A penile/urethral swab is taken from the male and is inoculated onto NYC agar and chocolate agar. The plates are incubated at 37˚C in CO

₂

at the clinic and later that evening are transported to the microbiology laboratory. A Gram stain is carried out on a smear of the penile discharge. The patient is also screened for other STI’s and is interviewed in relation to contact tracing his sexual partners.

Results

The following are the results obtained for the organisms growing on the chocolate agar and the organism growing on the NYC agar. Both agars were incubated in CO

₂

at 37˚C. Two organisms, A and B, were growing on the chocolate agar. Organism B was growing on both agars. This organism was identified as

Neisseria gonorrhoeae.

The preliminary identification of organism A was not obtained.

Neisseria gonorrhoeae

is the causative pathogen of gonorrhoeae, a sexually transmitted infection that is characterised by a pus filled infection of the surfaces of the mucous membranes of the throat, eye, vagina and urethra in males and females.  This pathogen can be spread through direct sexual contact or through vertical transmission from mother to baby during birth. Symptoms of this bacteria in males include painful urination and urethral discharge, while females present with increased vaginal discharge. Usually females infected with this pathogen present as asymptomatic and are the biggest reservoir of this STI (Edwards and Apicella, 2004).

Table 1:

Basic characterisation test results carried out for the chocolate agar plate and the NYC agar plate that were both incubated in CO

₂

at 37˚C for the 24 year old male patient in the STI Case Study 5. The preliminary identification of organism A was not obtained.

Plate name:	Chocolate agar plate incubated in CO 2 at 37˚C		NYC agar plate incubated in CO 2 at 37˚C	Controls: Positive	Negative
Test	Organism A	Organism B
Colonial morphology	0.5mm, grey, smooth, circular, convex, no odour.	0.5mm, light grey, smooth, circular, entire, no odour.	0.5mm, green, smooth, circular, entire, no odour.	/	/
Gram stain	Gram negative bacilli	Gram negative diplococci	Gram negative diplococci	/	/
Catalase	+	+	+	+	–
Oxidase	–	+	+	+	–
Preliminary identification	/	N eisseria gonorrhoeae	N eisseria gonorrhoeae	/	/

Legend:

Catalase

: + = positive for the enzyme catalase- bubbles produced.

–          = negative for the enzyme catalase -no bubbles produced

Oxidase

: + = positive for the enzyme oxidase – purple colour formed

–          = negative for the enzyme oxidase – no colour formed

Discussion

From the clinical details given in Case study 5 and from the basic characterisation tests, it is evident that the causative pathogen of the patient’s dysuria and penile discharge and the organism that was growing as organism B on chocolate agar and growing on the NYC agar is

Neisseria gonorrhoeae.

There are numerous further tests that could be carried out to confirm this causative pathogen

Neisseria gonorrhoeae

that the patient in this case study is infected with. This pathogen should be confirmed using two different methods of detection such as biochemical such as the API NH strip for

Neisseria

and

Haemophilus

species and molecular and serological testing. Such tests include the Nucleic Acid Hybridization Test (NAAT) that utilises a DNA probe that is labelled with a chemiluminescent tag and is targeted to a region of the 16s rRNA of  the

Neisseria gonorrhoeae

pathogen that is mixed with the patient’s sample. This assay is based on the hybridization of nucleic acids. In the patient’s sample if the pathogen is present, rRNA released from

Neisseria gonorrhoeae

will hybridize with the probe DNA. The probe that is not hybridized is removed. The DNA: RNA hybrids luminescence intensity is then measured. Samples used for this testing are endocervical and urethral swabs (Sood et al., 2014). According to the HPSC, NAAT testing is the standard test for the laboratory detection of

Neisseria gonorrhoeae.

The enzyme tube test, Gonocheck II can differentiate between the various

Neisseria

species such as

Neisseria meningitidis

,

Neisseria lactamica

and

Neisseria gonorrhoeae.

Specimens used for this test are well isolated colonies from either Modified Thayer Martin or chocolate agars.Enzymes produced by the bacteria act on colourless substrates to produce coloured end products.

Neisseria meningitidis

produces an end product that is yellow.

Neisseria gonorrhoeae

produces three enzymes – gammaglutamylaminopeptidase, hydroxyprolyaminopeptidase and betagalactosidase and produce a red-pink coloured end product, confirming this pathogen (CDC, 2018). The GeneXpert CT/NG System by Cepheid is a real time PCR NAAT platform that allows sample preparation, amplification and detection of

Neisseria gonorrhoeae

from patient urine samples, male urethral swabs and female vaginal and endocervical swabsin 90 minutes (Gaydos et al., 2013). The Abbott RealTime CT/NG utilises RT-PCR and a fluorescent labelled oligonucleotide probe that allows for direct, real-time, fluorescent, qualitative detection of the genomic DNA of

Neisseria gonorrhoeae

and plasmid DNA of

Chlamydia trachomatis

from patient urine samples, male urethral swabs and female vaginal and endocervical swabs (Gaydos et al., 2010).

Neisseria gonorrhoeae

possesses a wide abundance or virulence factors that enable it to efficiently establish infection and adapt to its hosts environment, as it did in this patient in the case study. The entry site of this bacteria in males is the urethral cells of the penis and the vagina in females. This pathogen mainly infects the epithelia of the urogenital tract and infects areas such as the rectal mucosa, pharynx and conjunctiva less commonly.

Neisseria gonorrhoeae,

with its repertoire of adhesion molecules attaches to the cuboidal and columnar epithelial cells present in the urethra, pharynx, endocervix and ano-rectal region. Such adherence molecules include pili, porin proteins – Opa and PI and type IV fimbriae. These adhesion molecules bind to host carcinoembryonic antigen cell adhesion molecules (CEACAM) receptors present on epithelial cells. Once attached to these receptors, the pathogen then rapidly proliferates and spreads up through the urethra in males and the cervix to the fallopian tubes in females where the infection and healing processes causes fibrosis, blockage and damage to the tubes. These adhesion molecules are able to evade being removed by vaginal discharge or urine. The pili and fimbriae facilitate attachment to the mucosal epithelium and the pili protein genes possess hypervariable and constant regions that enable the pathogen to exhibit antigenic variation by recombination of its surface antigens. This proves difficult in developing a vaccine for this bacteria and also for the production of host antibodies that are only effective for a short duration and so, are not protective against this bacteria. Pili also enable twitching motility that allows the bacteria to ascend the mucous lined surfaces (Edwards and Apicella, 2004). Porin protein (PI) is responsible for forming pores in the host cell membrane and induces apoptosis in the epithelial cells causing the shed of epithelial cells and fallopian tube damage in females. However, in a study carried out by using Chang epithelial cells, an anti-apoptotic role of porin proteins was hypothesized. It was found that enhancing the survival of epithelial cells of the urethra could allow the bacteria to multiply within an intracellular environment that is protected and thus, enhance the colonization of

Neisseria gonorrhoeae.

PI also allows the bacteria to survive following apoptosis.

Neisseria gonorrhoeae

also possesses a lipo-oligosaccharide layer (LOS) that exhibits endotoxin activity by inducing inflammation. Pelvic inflammatory disease that can result in fallopian tube infection and infertility is caused by the shedding of the LOS that initiates local inflammatory injury (Chen and Seifert, 2013). The LOS is able to evade the activation of the complement cascade by concealing itself with host sialic acid, rendering it unrecognisable by the host immune system. Opa proteins present on the surface of

Neisseria gonorrhoeae,

bind to the CEACAM family of adhesion receptors present on neutrophils, epithelial cells and B and T lymphocytes, facilitating the activation of the adaptive and innate immune responses upon epithelial cell infection (Sadarangani et al., 2010). TNF- α, a cytokine released during the host innate immune response is pro-inflammatory and has a profound damaging effect on the host epithelial cells such as the fallopian tubes, This cytokine prompts the production of phospholipases and proteases, inducing excess inflammation and damage.

Neisseria gonorrhoeae

contains the enzyme IgA protease at its core. This is responsible for breaking down the host IgA1 antibodies found in mucosal membranes that have an immune function in protecting against infections in the mucous membranes. This bacteria also possesses a capsule that allows it to resist opsonisation and phagocytosis as it similar in composition to that of the connective tissue of the host. Thus, this enables the bacteria to multiply, survive and spread within the host to carry out further infection and damage (Edwards and Apicella, 2004). All of these virulence factors culminated to initiate infection in the male patient in this case study to cause his burning and sore sensation while urinating and his purulent penile discharge. If gonorrhoeae is not treated, disseminated Gonococcal Infection (DGI) occurs. This is due to

Neisseria gonorrhoeae

spreading systemically to other parts of the body via the bloodstream, causing joint pain and arthritis and lesions on the skin and endocarditis may also result from DGI but this is rare. DGI is more common in females due to them more frequently being asymptomatic (Russ and Wrenn, 2005).

Further investigation that could be carried out for this patient includes contact tracing all of his previous sexual partners in the past two weeks and to notify them of his infection. The HPSC international guidelines for gonorrhoeae infections recommend that all male patients who have a urethral infection that is symptomatic must notify all of their sexual partners of the previous two weeks or if longer, their last partner This ensures that his previous sexual partners are made aware of his infection as they too may be infected and may not be displaying symptoms (asymptomatic). His previous partners will also undergo a full STI screen to establish whether they are infected with gonorrhoeae or other STIs. Contact tracing reduces transmission of

Neisseria gonorrhoeae

and its reinfection, while also informing and aiding individuals and healthcare workers in the understanding of the patterns of transmission within communities. In Ireland, gonorrhoeae is a notifiable disease under the Infectious Disease Regulations as this pathogen can have consequences later in life such as infertility (HPSC, 2018).

In 2017, there were 2249 notified cases of gonorrhoeae in Ireland, causing it to become the second most commonly encountered STI in Ireland. This pathogen has an incidence rate in Ireland of 47.2 per 100,000 population. However, these figures are believed to be underestimated as 55% of males and 86% of females suffering with

gonorrhoeae

infections are asymptomatic and so, the actual figures are believed to be a lot higher (HPSC, 2018).

References

• Cdc.gov. (2019). Enzyme Substrate Test – Gonorrhea – STD Information from CDC. [Online] Available at:
  
  https://www.cdc.gov/std/gonorrhea/lab/tests/enzyme.htm
  
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• Chen, A. and Seifert, H. (2013). Structure-Function Studies of the Neisseria gonorrhoeae Major Outer Membrane Porin.
  
  Infection and Immunity,
  
  [Online] 81(12), pp.4383-4391. Available at:
  
  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837997/
  
  [Accessed on: 3rd January 2019]
• Edwards, J. and Apicella, M. (2004). The Molecular Mechanisms Used by Neisseria gonorrhoeae To Initiate Infection Differ between Men and Women.
  
  Clinical Microbiology Reviews
  
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  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC523569/
  
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• Gaydos, C., Cartwright, C., Colaninno, P., Welsch, J., Holden, J., Ho, S., Webb, E., Anderson, C., Bertuzis, R., Zhang, L., Miller, T., Leckie, G., Abravaya, K. and Robinson, J. (2010). Performance of the Abbott RealTime CT/NG for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae.
  
  Journal of Clinical Microbiology
  
  , [Online] 48(9), pp.3236-3243. Available at:
  
  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937681/
  
  [Accessed on: 3rd January 2019]
• Gaydos, C., Van Der Pol, B., Jett-Goheen, M., Barnes, M., Quinn, N., Clark, C., Daniel, G., Dixon, P. and Hook, E. (2013). Performance of the Cepheid CT/NG Xpert Rapid PCR Test for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae.
  
  Journal of Clinical Microbiology
  
  , [Online] 51(6), pp.1666-1672. Available at:
  
  http://10.1128/JCM.03461-12
  
  [Accessed on: 4th January 2019]
• Hpsc.ie. (2017). Annual Epidemiological Report – Gonorrhoea in Ireland, 2017. [Online] Available at:
  

  Click to access Gonorrhoea%20in%20Ireland%202017(includes%20latest%20trends).pdf

  
  [Accessed on: 4
  
  ^th
  
  January 2019]

• Hpsc.ie. (2017). National Guidelines for the Prevention and Control of Gonorrhoea and for minimising the impact of Antimicrobial Resistance in Neisseria Gonorrhoea [Online] Available at:
  

  Click to access AMR%20Gonorrhoea%20guidelines%20documetn%20FINAL%202017.pdf

  
  [Accessed on: 4th January 2019]

• Russ, S. and Wrenn, K. (2005). Disseminated Gonococcal Infection.
  
  New England Journal of Medicine
  
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  https://www.nejm.org/doi/full/10.1056/NEJMicm040620
  
  [Accessed on: 2nd January 2019]
• Sadarangani, M., Pollard, A. and Gray-Owen, S. (2011). Opa proteins and CEACAMs: pathways of immune engagement for pathogenicNeisseria.
  
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• Sood, S., Verma, R., Mir, S. S., Agarwal, M., Singh, N., Kar, H. K., & Sharma, V. K. (2014). Nucleic acid amplification tests (NAATs) for gonorrhoea diagnosis in women: experience of a tertiary care hospital in north India.
  
  The Indian journal of medical research
  
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  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311319/
  
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Practical Risk Assessment Form

Hazard	High	Medium	Low	Current control measures for this hazard	Options for improved controls
Biological: Neisseria gonorrhoeae Oxidase positive and negative controls Catalase positive and negative controls		X X X		Good aseptic techniques to control the exposure of this infectious agent. Using gloves and laboratory coat to avoid becoming contaminated with these controls and bacteria. Using disinfectant to clean the surface after working with these controls and the bacteria. Disposing gloves, slides and agar plates when they are finished with.	Avoid touching the face or mouth with gloved hands to prevent becoming contaminated with the controls and bacteria.
Chemical: Gram stains – Crystal Violet, Gram’s iodine, acetone and Carbol Fuschin Hydrogen peroxide			X X	Wearing gloves and laboratory coat to protect hands, clothes and skin from being stained with these chemical agents. Wearing closed footwear rather than sandals or open-toed shoes. Avoid inhaling or ingesting the chemicals.	Closing laboratory coat properly. Wearing safety glasses.
Electrical: Microscope Hot plate		X X		Correct handling of the microscope. Avoid tangling the leads and do not have them hanging down on the floor. Turning off the equipment when they are not in use	Not leaving them at the edge of the benchtop.
Physical: Glass slides Chairs		X	X	Proper handling of slides. Disposing of them in the sharps bin when they are finished with. Keeping chairs pushed in under the workbenches.	Not dropping slides or disposing of them in the biohazard bins.