Neisseria gonorrhoeae

• Etiology of Gonorrhea
• Greek (gonos – seed and rhoia- flow)
• First described in urethral discharge by Neisser in 1879
• Cultured by Bumm in 1985 and proved its pathogenecity by inoculating human volunteers
• Resemble meningococci very closely in many properties

Sample:

• Endocervical swab (not vaginal swabs)
•   Uretheral discharge,  urine (in males)
•   Discharge after prosthetic massage,
•   Conjunctival swab in case of opthalmia neonatrum
• – Throat infection may also occurs, in such case throat swab
• In DGI, specimen may include blood, swabs of skin lesions, urine
  

  ---

Sample collection

(A)Collection or urethral discharge from male:

• Cleanse around the urethral opening using a swab moistened with sterile physiological saline.
• Gently massage the urethra from downwards, using a swab, collect a sample of discharge.
• Make a smear of the discharge on a microscope slide by gently rolling the swab on slide which will avoid damaging pus cells that contain the bacteria.
• Note: Very few pus cells may be present if the patient has recently passed urine. Allow 2–4 hours after urination before collecting a specimen.
• When culture is indicated, collect a sample of pus on a sterile cotton-wool swab.
• To isolate from urine, early morning first voided urine sample is taken, centrifuge and take the sediments.
• A rectal swab is required in case of homosexual patients.

(B) Collection of cervical specimens from female

• Use a sterile vaginal speculum to examine the cervix and collect the specimen.
• Moisten the speculum with sterile warm water, and pass so that cervix could be visualized properly. Do not lubricate the speculum with a gel that may be bactericidal.
• Cleanse the cervix using a swab moistened with sterile physiological saline.
• Pass a sterile cotton-wool swab 20–30 mm into the endocervical canal and gently rotate the swab against the endocervical wall.

• ---

Transport media:

• Spot inoculation of sample on culture medium

If not possible,

• Specimens should be collected on charcoal impregnated swab
• –Transported to lab on Stuart’s transport medium

Gram Staining: Gram negative, bean shaped diplococcus, intracellular as well as extra cellular

• Characteristic  intracellular gram negative diplococci  lying with polymorphoinuclear leukocytes and few extracellular  – positive
• Aprrox. 95 % infected male yield positive result
• Not reliable in women unless intracellular gram negative diplococci is seen as there are other bacteria with similar morpholgy which appear like Neisseria gonorrhoeae

Direct detection:

• Direct detection in patient secretion can be done which avoids problems with transport.
• Gonozyme test: polyclonal antibody test to detect gonococcal antigen by enzyme immunoassay.
• Direct florescence test
• Slide agglutination test

Culture the specimen

• Modified New York City (MNYC)
• Thayer Martin medium with VCN
• Chocolate agar

-Incubation at 37°C for 24 hour in a moist atmosphere containing 5-10% CO2 by placing a damp piece of filter paper at the bottom of the jar.

ADDITIONAL CULTURE

– Inoculate the specimen on two plates of Blood agar and incubate one anaerobically and the other aerobically at 35–37°C overnight.

Colony characteristics:

• small, round, translucent, convex colonies grow with finely granular surface after 24 hrs
• After 48 hrs, colonies are larger with crenated margin and opaque raised center
• On Thayer Martin medium, growth is slow

Kellogg divided gonococci into 4 types on the basis of coloial morphology, auto-agglutinability and virulence

Four types of colonies

–  T1, T2: virulent and contains pilli, small brown colonies

–  T3, T4: avirulent and nonpiliated, large granular, non pigmennted

-Test suspected colonies by touching with a cotton  bud soaked in oxidase reagent, contact area of bud turns purple within 5-15 Secs.

Biochemical reaction

• Catalase ,Oxidase, Glucose: +ve
•  Lactose, sucrose and maltose: -ve

Antibody detection in patients sera:

• Complement Fixation test (CFT)

Beta lactamase testing:

•  Inoculate the medium with several colonies of organisms and place 6 microgram of penicilln disc on the well, if ZOI is less than 20 mm after overnight incubation, test for beta lactamase by chromogenic cephalosporin method
• -Nitocephin, a chromogenic cephalosporin is normally yellow and turns red when hydrolyzed.
• -Inoculate 50µl of heavy suspension of bacteria into the well of a microtitre plate and add 10µl of nitrocephin solution
• -Incubate 30 mins, observe for color change.
  

  ---

Sensitivity to physical and chemical agents 

• Verile fragile organism
• Readily killed by drying, soap and water and many other cleaning or antiseptic agents
• May remain viable for a day in pus contaminating linen or fabrics
• Cutured gonococci dies in 3-4 days at room temperature
• Stored for several months by harvesting the isolate into 1 ml trypone soya broth containing 6% lactose at -20°C or -70°C

Pathogenesis

• Venereal disease transmitted by sexual contact
• Incubation peroiod – 2-8 days
• Confined to mucus secreting epithelial cells
• Pili mediates the adhesion og gonococci to the urethra or mucosal surface
• Cocci penetrates through the intercellular space and reach subepithelial connective tissue by the 3rd day after infection
• Penetrate columnar epithelial cells. Stratified squamous epithelium is resistant
• Attack mucous membrane of genitourinary tract, eye, rectum and throat producing acute suppuration followed by chronic inflammation and fibrosis

Disease in men

• Acute urethritis (most common clinical presentation in male few days after unprotected sexual intercourse)
• Dysuria and purulent penile discharge
• Infection may extend to prostate, seminal vessicle and epididymus
• Chronic urethritis may lead to stricture formation
• Infection may spread to periurethral tissue causing abcesses and multiple draining sinuses

Disease in woman

• Endocervix is the primary site of infection and extends to urethra and vagina giving rise to mucopurulent discharge
• Vaginal epithelium are resistant to infection in adults but severe vulvovaginitis may occur in prepubertal girls
• Asymptomatic carriage in woman is common (rare in man)
• Symtomatic patients commonly experience vaginal discharge, dysuria and abdominal pain

Non-gonococcal urethritis in men

• Chlamydia trachomatis is a common cause of non-gonococcal urethritis (NGU), particularly in men.
• A presumptive diagnosis of NGU can be made when a urethral smear contains 5 or more pus cells and no intracellular Gram negative diplococci (or more than 15 pus cells in a first voided urine specimen from a male patient).