Valery Taustsiakou
G00296946
30th November 2015
Co-workers: Aishlinn Jennings, Veronica Gomes.

Urinary Tract Cases and STI cases

UTI/ Case 2
Clinical details: * 20year old female presented to student health clinic with characteristic UTI symptoms. * Her symptoms were dysuria, frequent and painful micturition for previous 2 days. * It was reported that she has not observed any blood in her urine. * Her temperature was checked and was normal. * An absence of flank pain was reported. * A mid-stream urine sample (MSU) sample was collected and sent to the microbiology laboratory for investigation. * The specimen was cultured on chromogenic UTI agar and CLED agar. * A diagnosis of UTI or cystits was made at the student health clinic. * This diagnosis requires laboratory confirmation.

Results:

Table 1. White cell count (WCC), red cell count (RCC) and bacterial colony count results from patient’s midstream urine sample. Parameter | Number counted | Count (no./mm) | Reported/cmm | units (CFU/ul) | units (CFU/ml) | White cell count (WCC) | 374 | 374,000 | >100WCC/cmm | - | - | Red cell count (RCC) | 27 | 2,700 | Present | - | - | Bacterial colony count | 200* | - | - | - | 2x105* |

*= Macroscopic bacterial colony count was performed by the supervisor within a mixed but sparse colonial growth on the Orientation Chromagar medium.

The macroscopic appearance of the urine samples was a cloudy turbid fluid.
One epithelial cell was observed within the microscopic WCC and RCC.

Table 2. Morphological and preliminary identification of UTI pathogen from MSU cultured sample.

Characteristic | Chromogenic UTI agar | CLED agar with Andrades indicator | Colonial morphology | Purple raised 1.5-3mm shiny umbonate colonies with a purple periphery**. | 1. Moderately sized 2mm Round, entire, creamy, blue opaque slightly raised colonies*.2. small-moderately sized 1-2mm colonies.Lactose fermenting orange mucoid colonies on pink agar. Umbonate, translucent, entire, round, shiny, slightly raised colonies. | Gram stain | Gram positive cocci | 1. NP2. Gram positive cocci | Catalase | positive | 1. NP2. positive | Coagulase | negative | 1. NP2. negative | Oxidase | NP | 1. NP2. NP | Preliminary ID: | Staphylococcus saprophyticus | 1. NP2. Staphylococcussaprophyticus |

Two colony types with distinctive colonial morphologies were observed only on CLED agar.
-NP= Not performed.

*= These colonies were considered typical skin contaminants and were therefore not tested.

**= There was a mixed growth of colonies on the chromogenic UTI agar including similar colonies that looked like skin contaminants.

Discussion:
The patient was a 20 year old young female who presented to the student health clinic. She had characteristic symptoms of a urinary tract infection (UTI), complaining of excessively frequent and painful urination. She did not have any flank pain suggesting that she did not have pelvic inflammatory disease (PID). She did not report any blood in her urine. Therefore her symptoms suggest the UTI was uncomplicated. In complicated UTI, patients typically present with abdominal pain if the UTI is sufficiently deep and in the bladder (Ray, Ryan and Sherris, 2014).

Macroscopic urine sample appearance and Microscopic count:
Macroscopically the urine sample was observed as being turbid, cloudy and white. A normal urine sample would typically be straw-yellow coloured in appearance. Her urine did not appear red-coloured, but appeared cloudy to indicate the presence of pus, white cells and a possible infection. The bacterial colonies counted on the Orientation Chromagar were counted from a mixed growth culture plate. Routinely this mixed growth may be stated in the patient’s report. If the colony count cannot be performed as a result of excess and mixedcolonies, a repeat midstream urine sample would be requested.

The white cell count was 3,740WCC/mm (Table 1) greater than the normal reference range. This result is significantly greater than the normal reference range of 10 WCC/cmm in urine which is one of the parameters indicative of a UTI (Najar, Saldanha and Banday, 2009). However, to diagnose UTI, this result must be complemented by a significant colony count of pure growth as in the UK investigation guidelines (No author, 2014). The patient’s colony count was 2X105 colony forming units (CFUs) /ml of urine (Table 1). In order to diagnose UTI, the colony count must be greater than 100,000 CFUs/ ml (Najar, Saldanha and Banday, 2009). The combined parameters of an excessive colony count and pyuria in the patient’s urine confirm the laboratory diagnosis of a UTI.

The results suggested that white cells migrated to her urinary system to combat a UTI infection. The white cells particularly migrated to her urethra region to destroy the infection causing cystitis. There was moderate number of red cells counted in her urine sample. However her urine sample was not bloody when observed macroscopically. This was suggestive of a bleed from her urethra or shedding of red blood cells from a recent menstrual period (No author 2014). However this shedding was not significant in her case and most likely did not indicate an internal bleed. A true internal bleed caused by a UTI pathogen would be indicated by a red-coloured urine. Urine passes through the urinary system including the urethra.

Colonial morphology and preliminary identification:
From Table 2, the colonial morphology of the colonies denoted by the number 2 on CLED agar with andrades indicator, have similar colonial morphology as the predominant organism that grew on Orientation Chromagar agar. The yellow colonies on CLED were lactose fermenters. The colonial type observed on CLED and Oxoid chromogenic agars was indicative of the same pathogenic organism.
A mixture of growth on the chromogenic agar plate was observed and it was difficult to distinguish between the different colony types. The pathogenic organism was isolated on its own and was distinctive by its biochemical testing. The gram stain was the same for both the colonies tested from the different agars. The biochemical tests of the colonies experimented on from both agars were similar. Therefore the predominant organism on both agars with similar colonial morphology was assumed to be the causative agent of UTI.

Gram positive cocci were microscopically observed in clusters. The gram stain and the colonial morphology prompted biochemical testing for catalase and coagulase. Staphylococcus spp. can be a cause of UTI infections. The similar colonies on CLED and Oxoid chromogenic agar plate were tested biochemically to prove they were the same micro-organism. The colonies tested catalase positive and coagulase negative -indicative of coagulase-negative staphylococci (CoNS).
The positive catalase test indicated that this was a Staphylococcus pathogen. The catalase test was positive because catalase was produced by the micro-organism. Released catalase caused the effervescence reaction, by catalysing the hydrogen peroxide test reagent.
The slide coagulase test was negative because the plasma reagent did not clump as a result of the lack of coagulase enzyme. This result indicated that the pathogenic organism was not Staphylococcus aureus. The coagulase test suggested that the organism was a CoNS. To completely exclude the possibility of S. aureus a Staphylase kit test was performed. This test, which detects bound coagulase, was negative. Therefore the organism was most likely a CoNS. The specific CoNS species still requires confirmation. The lactose fermentation on CLED agar supports the possibility that this CoNS could be Staphylococcus saprophyticus.

Confirmatory testing was not performed but the novobiocin test differentiates between two species of CoNS including Staphylococcus epidermidis and S. saprophyticus. The novobiocin test is an antibiotic susceptibility test performed by placing novobiocin antibiotic onto an agar plate. The organism is cultured and, if susceptible, a zone of lysis is observed around the novobiocin disk. S. epidermidis is susceptible to Novobiocin while S. saprophyticus is resistant (No author, 2015).

Pathogenesis and virulence factors:
The gastrointestinal tract is a major reservoir of S. saprophyticus (Raz, Colodner and Kunin, 2005). A study found that 4.6% of women aged 13-40 years had S. saporphyticus isolated from their genital tract. This indicated that S. saprophyticus is part of the normal flora (Raz, Colodner and Kunin, 2005).
S. saprophyticus virulence factors permit the dissemination of the organism. The organism is a pathogen in the urinary tract if it becomes displaced into the urinary tract. This pathogen is common in sexually active females (Raz, Colodner and Kunin, 2005).
Surface-associated lipoteichoic acids mediate the adherence of S. saprophyticus to urothelial cells. This adhering action permits the organism to resist the flushing action of urine. S. saprophyticus has a haemaglutinin that permits binding to fibronectin, a structural component of urothelial cells (Raz,Colodner and Kunin, 2005). S.saprophyticus also has a haemolysin and an extracellular slime layer. The extracellular slime layer permits the organism to attach to urinary catheters and other mechanical devices such as grafts, prosthetic valves, prosthetic joints and shunts (Ryan, Ray and Sherris, 2014). A catheter is a mechanical urine collecting device and requires proper sterilisation. Organisms with a slime layer typically cause problems in the insertion of mechanical devices especially when the sterility and innate skin barrier has been compromised (Ryan, Ray and Sherris, 2014). The slime layer in S. saprophyticus is a loose, bonded water-soluble firm layer. It consists of monosaccharides, proteins and peptides to mediate binding to tissues such as urothelial cells. (Ryan, Ray and Sherris, 2014). The protein receptors to which this organism binds in the urothelial tract have not yet been investigated.

Age and gender of the patient:
The female patient is 20 years of age and she is therefore sexually mature. UTI caused by S. saprophyticus commonly occurs in young sexually active women when it is displaced during sexual intercourse (Raz, Colodner and Kunin, 2005). It was suggested in the report written by by Raz and colleagues that condoms are a source of spermicides, thereby resulting in disruption of vaginal normal flora. Spermicides have a strong association with the occurrence of UTI. Therefore, condom use could have altered the normal vaginal flora, to permit colonisation of her urinary tract by S. saprophyticus.

STI/Case 5:
Clinical details: * 24 year old man presented to the sexually transmitted infection (STI) clinic. * He reported characteristic STI symptoms consisting of painful, burning urination and greenish-yellow mucopurulent discharge. * He stated that he had unprotected sex with several partners in the previous two weeks. * Urethral/penile swab was inoculated onto Modified Thayer Martin medium and New York City (NYC) agar. * Both agars were incubated at 37°C in CO2.

Table 1. Morphological and preliminary identification test results of STI causing pathogen. Characteristic | Modified Thayer Martin (MTM) agar | NYC agar | Colonial morphology | Round, entire, creamy white, opaque 3mm diameter golden white and slightly raised colonies. | No growth observed* | Gram stain | Gram negative diplococci. | - | Catalase | positive | - | Coagulase | -NP | - | Oxidase | positive | - | Preliminary ID: | Neisseria gonorrhoea | - |
Note: - = Not performed.

.

Discussion:

Gram stain:
A category type 2 Neisserial species was provided. Neisseria gonorrhoeae (N. gonorrhoeae) is a category type 3 pathogen and the college laboratory did not have a category 3 containment room to investigate this pathogenic organism. Also the lack of growth on New York City (NYC) medium was observed. NYC medium contains selective agents specifically for the growth of N. gonorrhoeae. The selective agents are mainly antibiotics such as vitamin B12 and Colistin to inhibit gram negative bacterial growth. Other selective agents and antimicrobial agents are present in the NYC agar.

There was a problem encountered in the category type 2 Neisserial species in terms of its gram stain. The gram stain did not appear as gram negative diplococci intracellular organisms. If N. gonorrohoeae was the pathogen investigated then the organism would be observed as gram negative diplococci. The biochemical tests were tested catalase and oxidase positive characteristic of N. gonorrhoea.

N. gonorrhoeae require an aerobic incubation supplemented with a carbon dioxide environment in 37’C for effective growth. All Neisseria are biochemically oxidase positive because they require an aerobic environment for growth on enriched medium (Ryan, Ray and Sherris, 2014).

Further confirmatory tests: N. gonorrhoeae is confirmed by biochemical sugar tests:
N. gonoorhoea can be identified with the Vitek-2 NH colorimetric biochemical testing cards.
N.gonorrhoeae catalyses glucose but not maltose, lactose or sucrose (Valenza et. al. 2007).
The GonoGen II is a monoclonal antibody (mAb) based test for the identification of N. gonorrhoeae from culture. In that test a buffer solubilises the cell wall to strip the cell wall. The outer membrane proteins become exposed. The outer membrane proteins contain the species specific antigens. The mAbs are linked to a red metal sol-particle which bind to the antigens and produce a colour. Therefore the test is a colorimetric agglutination biochemical test. There is another biochemical test known as the Genochek II test. The Genochek II is an enzymatic tube test for the differentiation of Neisserial species and Moraxella catarrhalis since Moraxella also has the characteristic gram negative diplococcus gram stain. The enzymes produced by the species are detected by production of chromogens from colourless substrates. N. gonorrhoeae produces a red-pink colour in this test.
There are several molecular assays available for the detection of N. gonorrhoea. The NUCLEIC ACID HYBRIDISATION ASSAY DNA PROBE (NAHADP) hybridises nucleic acid. A chemiluminescent DNA probe targets a portion of 16S rRNA of N. gonorrhoea within the patient sample. rRNA released from N. gonorrhoea binds to the probe DNA. The non-hybridised probe is removed by washing. The intensity of the DNA/RNA hybrid is measured to observe if this hybrid nucleic acid bound complex is above the detection threshold. The NAHADP assay uses urethral and endocervial swab specimens.
Abbot Realtime Chlamydia trachomatis (CT)/ N. gonorrhoea (NG) test is based on a fluorescent labelled oligonucleotide probe that targets specific DNA regions for amplification. Nucleic acid Amplification tests (NAAT) consist of different assays based on PCR, ligase chain reaction (LCR), strand-displacement assay (SDA), hybrid capture assay (HCA) and transcription mediated assay (TMA). All the NAAT assays show equal or higher sensitivity compared to culture (Ng and Martin 2005). All the NAAT assay are highly specific at >99.6% specificity. It is recommended by the CDC that N. gonorrhoea is identified by two different methods before a confirmation of the infectious pathogen is made.
Matrix assisted laser desorption ionisation and time of flight (MADLI-TOF) has been developed for N. gonorrhoea testing (Carranente et. al., 2015). MALDITOF still requires research work to update strains of N. gonorrhoea. MALDITOF is beneficial for epidemiological typing because of its ability to differentiate many species of N. gonorrhoea.

Virulence factors, Pathogenesis of pathogen and further investigation of this patient:
N. gonorrhoea has structural components consisting of cell surface components and outer membrane proteins. The cells surface components consist of an antiphagocytic capsule, and pili, The PilE is an antigenically variable pilus. The pilli mediate attachment of the gonococci to epithelial cells. The outer membrane proteins consist of Por I (Protien I), Opa (Portein II), Rmp (Protein III), transferrin binding proteins, a lactoferrin binding protein and LOS (lipooligosaccharide). N. gonorrhoea produces an IgA1 protease extracellular product. N. gonorrhoea does not produce any exotoxins but produces the endotoxin LPS because it is a gram negative organism. Gram negative organisms have LPS as their typical characteristic feature (No author, 2015).

The pathogenesis of N. gonorrorhoeae has three stages. The sexually transmitted pathogen attaches and invades mucosal tissue, survives in the submucosa and disseminates. The organism enters the urinary tract by sexual contact by vaginal or anal transmission. The organism reservoir is the sexual reproductive fluid of an infected individual. The organisms is transmitted in the sexual fluid by infected individuals (Ryan, Ray and Sherris. 2014).

The pathogen is introduced onto a mucosal surface by sexual contact with an infected individual. Pili and Opa-proteins function as adherence ligands. The pillus is a ligand of immune recognition and therefore the pillus subunits are varied to avoid immune recognition. Pilli and Opa proteins initially attach to receptors CD46, CD66 and integrins on non-ciliated epithelial cells. Initial adherence is controlled by the pilli which have proven to generate movement across the cell surface (Ryan, Ray and Sherris. 2014). The Opa proteins establish a tighter attachment to prevent displacement off the mucosal tissue, by urinary flushing. The outer membrane proteins (OMPs) establish a route for pathogen entry to invade the urothelial cells. The OMP Por1A stimulates host enzyme systems. This stimulation triggers phagocytosis of the gonococci using microfilaments and microtubules of the urothelial cells (Ryan, Ray and Sherris. 2014). Another route of entry inside the urothelial cells is the association of reduction-modifiable proteins (Rmp) with with Por protein to form pores in the cell surface of the colonised columnar epithelial cells (No author, 2015). Por B is a major OMP that prevents neutrophil degranulation and phagosome formation. Approximately 60% of N. gonorrhoea strains have porB on their cell surface (Ryan, Ray and Sherris, 2014). Inhibiting phagosome formation prevents killing of N. gonorrhoea by the host immune system and permits N. gonorrhoea to thrive intracellularly (Male et al., 2012).

The gonococcus induced the host cell to actively pull or draw in the pathogen internally. Once inside the bacteria transmigrates the cell to exit into the basal membrane into the submucosa, effectively bypassing the mucosal cell layer. Therefore the rapid access past mucosal tissue induced an acute infectious response within the patient after the organism proliferates. The patient had a rapid infectious symptoms of an STI in the previous two weeks. It is not known if the STI appeared within 2-7 days as in this patient case

Attachment and invasion are divided into their stages of adherence, phagocytosis and trancellular migration. The pilli and Opa proteins are responsible for the attachment to nonciliated epithelial cells. The gonococci stimulate the host to perform phagocytosis. The host internalises the pathogen by host microtubules to enter into the cells. This is known as endocytosis. The bacteria pass into the submucosa and disseminate (Ryan, Ray and Sherris. 2014).

The superficial defences of the genital mucosa are overcome by the extracellular secretion of IgA1 protease by N. gonorrhoeae. The IgA1 protease molecule cleaves the hinge region of IgA antibodies. IgA is secreted into the mucosal tissue as a protective immune feature by the host. The mucosal tissue is left unprotected if IgA is degraded, and the pathogenic organism colonises the musical tissue. The pillus E (pilE) cell surface component of N. gonorrhoeae varies antigenically to prevent immune recognition. PilE is a major antigen varied cell surface protein. It permits the stealthy invasion of the mucosal tissue, undetected by toll-like receptors (Ryan, Ray and Sherris. 2014).

N. gonorrhoea survives in the submucosa by uptaking iron, blocking complement system deposition and interfering with phagocytosis. N. gonorrhoea has siderophores on its surface which are iron uptake receptors. These receptors interact with human iron transport proteins to scavenge the host’s iron. N. gonorrhoea syliates its lipo-oligosaccharide (LOS) by incorporating host sialic acid onto its own surface. The silyated LOS can then bind factor H of the alternative complement system to prevent C3b opsonin complement deposition. Therefore the bacterium cannot be targeted for destruction by phagocytes. Phagocytes like neutrophils and macrophages require opsonins to tag the surface of a bacterium for immune degradation.
Transferrin-binding proteins are not part of the siderphore system. They mediate the acquisition of iron. (Noinaj, Buchanan and Cornelissen, 2012). Lactoferrin-binding proteins acquire iron from lactoferrin (No author, 2015).

The cell surface components pilli and Opa proteins are antiphagocytic (Ryan, Ray and Sherris. 2014). The N. gonorrhoeae has the ability to resist oxidative killing by phagocytes. N. gonorrhoeae produces vast quantity of catalase to degrade oxygen radicals secreted by phagocytes. This characteristic feature is useful for the catalase biochemical test to identify the from this patient. To conclude N. gonorrhoea’s virulence components prolong its survival in the submucosa, by retarding neutrophil killing (Ryan, Ray and Sherris. 2014).

In the dissemination phase N. gonorrhoea tends to remain local to genital structures and cause inflammation. The peptidoglycan and LOS cause shedding and local injury continuing the pathogen’s proliferation. The purulent exudates of gonococci clusters are dry and sticky held together by Opa proteins. The patient had a mucopurulent penile discharge consisting of green-yellow exudate containing clusters of gonococci. (Ryan, Ray and Sherris. 2014). This indicated that the pathogen has multiplied and is in the dissemination phase. The patient was therefore symptomatic as a result of the pathogen having progressed to the dissemination stage. Infection may migrate deeper by extending Opa proteins while twitching by pili movement (Eriksson et. al., 2015). The cell surface components can extend and bind adjacent mucosal tissue. As more mucosal covered cells are invaded more iron can be utilised to facilitate cell division. Iron is an essential DNA replication component and contributes greatly in the dissemination phase. N. gonorrhoea in men migrates to the prostate and epididymis. N. gonorrhoea migrates to the paracervical glands and fallopian tubes in women (Edwards and Apicella 2004). N. gonorhoeae releases peptidoglycan during growth causing harm to the ciliated epithelium of the fallopian tube. The main consequences of a late diagnosis in patients are infertility and further spread to their sexual partners. Therefore it was important for the patient to receive their diagnosis and treatment early. Damage to the genitals by LOS, lipopolysaccharide (LPS) and peptidoglycan impairs fertility N. gonorrhoea does not produce toxins except for endotoxin production (Murray, Rosenthal and Pfaller, 2012). Lipid A is the endotoxic component of LPS exerting toxic effects.
N. gonorrhoeae is suggested to be an intracellular pathogen by surviving in dead phagocytes. However this in-vitro finding still must be proven by additional in-vitro experiments. Its mechanism of survival are still not proven in the research literature (No author, 2015).

Incidence of Neisseria gonorrhoea in Ireland:
The highest crude incidence rate (CIR) was in HSE east at 51/100,000 in 2013. Dublin, Waterford and Sligo were the counties recorded with the highest incidence rates in 2013. Therefore public sexual health campaigns and screening would be effective on the Dublin, Waterford and Sligo populations.

Dublin had an incidence of 59.2/100,000 higher than the incidence rate for HSE east. However HSE east incidence statistics also consist of screening Counties such as Meath, Louth and Offaly. Therefore Dublin is the focal point of N. gonorrhoea transmission and diagnosis. Waterford and Sligo have crude incidence rates of 33.4/100,000 and 27.5/100,000 respectively. The Dublin population may have an increased risk of risky sexual activity contributed by the varied socio-demographics and cultural diversity.

Galway County had a crude incidence rate of 10-20/ 100,000 recorded in 2013. Rates should be interpreted with caution since the data was collected by positioned clinics and not by patient address. Therefore there most likely were N. gonorrhoeae individuals missed during the gathering of statistical data and screening.

Within the document it was reported that there was a 3-4 fold rise in gender incidence of N. gonorrhoea from 2009-2013. The HSE west and HSE Midwest did not record a rise in the CIR. Other HSE regions experienced an increased incidence rate of N. gonorrhoea. The CIR rates were gathered by clinics set up across the country. The public awareness campaigns may not have been as rigorous in rural areas of counties compared to dense urban areas. In dense urban areas there would have seen a higher number of participants. The higher number of participants skewed the average participation in other counties. Therefore the combined CRI for HSE east region would not be fully representative of less centralised counties. Therefore the illustration of Ireland with incidence rates were very interesting and useful to demonstrate the counties with the highest incidence rates.

Risk assessment:

Hazard: | high | normal | low | control measures: | Biological: Neisseria gonorrhoea is a category group 3 pathogen. Urine count on patient case 2 was performed. Staphylococcus saprophyticus was a category 2 pathogen. | | * | | A different category type 2 Neisseria species was provided by the supervisor.Latex gloves and a white Howie laboratory coat was worn.Latex gloves were worn to protect against the potentially biological hazardous urine sample. If N. gonnorhoeae were to be investigated in a category 3 biocabinet, latex gloves would prevent contact with the infectious urethral/penile swab. The filtration system of the biosafety cabinet would filter the pathogen. | Chemical:Hydrogen peroxideCoagulase reagent.Staphylase reagent-animal origin.Gram stain reagents: Crystal violet, Grams Iodine, Acetone, Carbol Fuchsin, | | | * | Latex gloves are impermeable fluids but may not be impermeable to gram stain reagents. Precaution and care was implemented with gram stain reagents because gram stain reagents are concentrated chemicals. Personal protective clothing and latex gloves were sufficient. | Electrical: Microscope has electrical components. | | | * | Gram stain reagents were kept away from the microscope. Chemical testing reagents for catalase and coagulase also kept away from the microscope. | Physical: Backpacks. Proper posture during microscopic evaluation. | | | * | Moved backpacks into a corner of the laboratory and, not underneath the workbenches. A proper posture was essential during microscopy evaluation in order to prevent back injury. |

References:

UTI

Najar, M., Saldanha, C. and Banday, K. (2009). Approach to urinary tract infections. Indian J Nephrol, 19(4),129.

No author, 2014. UK Standards for Microbiology Investigations Investigation of Urine, Standards Unit, Microbiology Services, PHE, NHS. Bacteriology, 7.2. 1-46.
Available at: https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/458681/B_41i7.2.pdf
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No author, 2015. Novobiocin Differentiation Disks. Hardy Diagnostics.
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Raz, R., Colodner, R. and Kunin, C. (2005). Who Are You Staphylococcus saprophyticus?. Clinical Infectious Diseases, 40(6), pp.896-898.

Ryan, K., Ray, C. and Sherris, J. (2014). Sherris medical microbiology 6th ed. New York: McGraw Hill Medical.

STI:

Carannante, A., De Carolis, E., Vacca, P., Vella, A., Vocale, C., De Francesco, M., Cusini, M., Del Re, S., Dal Conte, I., Cristaudo, A., Ober, P., Sanguinetti, M. and Stefanelli, P. (2015). Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for identification and clustering of Neisseria gonorrhoeae. BMC Microbiology, 15(1).

Edwards, J. and Apicella, M. (2004). The Molecular Mechanisms Used by Neisseria gonorrhoeae To Initiate Infection Differ between Men and Women. Clinical Microbiology Reviews, 17(4), 965-981.

Eriksson, J., Eriksson, O., Maudsdotter, L., Palm, O., Engman, J., Sarkissian, T., Aro, H., Wallin, M. and Jonsson, A. (2015). Characterization of motility and piliation in pathogenic Neisseria. BMC Microbiology, 15(1).

Male, D., Brostoff, J., Roth, D. and Roitt, I. (2012). Immunology. London: Elsevier Health Sciences UK. Murray, P., Rosenthal, K. and Pfaller, M. (2012). Medical Microbiology. London: Elsevier Health Sciences. Ng, L. & Martin, I.E. 2005. The laboratory diagnosis of Neisseria gonorrhoea. The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie médicale / AMMI Canada, vol. 16, 1, 15-25.

Noinaj, N., Buchanan, S. and Cornelissen, C. (2012). The transferrin-iron import system from pathogenic Neisseria species. Molecular Microbiology, 86(2), 246-257.

No author, 2015. Neisseria gonorrhoeae (“The Gonococcus”)– Gonorrhea. 45644: Medical microbiology [online].
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Valenza, G., Ruoff, C., Vogel, U., Frosch, M. and Abele-Horn, M. (2007). Microbiological Evaluation of the New VITEK 2 Neisseria-Haemophilus Identification Card. Journal of Clinical Microbiology, 45(11),3493-3497.

2013. Gonorrhoea in Ireland, 2013. HSE.
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