Neisseria gonorrhoeae lecture iii term

Morphology,pathogenicity,laboratory diagnosis

1. Neisseria gonorrhoeae A Lecture By – Dr. D. W. DESHKAR ASSISTANT PROFESSOR DEPT.OF MICROBIOLOGY D.Y.PATIL MEDICAL COLLEGE,KOLHAPUR D.Y.PATIL EDUCATION SOCIETY INSTTUTION DEEMED TO BE UNIVERSITY

2. CLINICAL CASE 2 A 20 year old man presented with urethral discharge & dysuria for previous two days. H/O unprotected sex with a commercial sex worker a week back. On examination of a smear of pus – Gram negative diplococci were seen inside polymorphs. Culture on Thayer – Martin medium - Positive Diagnosis – Gonorrhoea

3. Neisseria gonorrhoeae 3 N.gonorrhoeae causes the venereal disease gonorrhoea. Gonococcus was first described in gonorrheal pus by Neisser in 1879. Bumm in 1885 cultured the coccus & proved its pathogenicity by inoculating human volunteers.

4. Neisseria gonorrhoeae 4 The name Gonorrhea is derived from Greek words- Gonos ( seed ) rhoia ( flow ). Describes a condition in which semen flowed from the male organ without erection,

5. MORPHOLOGY Gram – ve diplococcus. With adjacent sides concave (kidney shaped) Predominantly in the polymorphs ( Intracellular) Some cells may contain as many as 100 cocci.

6. MORPHOLOGY Gonococci possess pilli on their surface. Pilli facilitate adhesion of the cocci to mucosal surface & promote virulence by inhibiting phagocytosis. Gonococci infect non – cornified epithelium. They agglutinate human RBCs. Haemagglutination not inhibited by mannose.

7. CULTURAL CHARACTERS Gonococci are non motile. More difficult to grow. Are aerobic. But may grow anaerobically also. Growth occurring best at pH 7.2 – 7.6 Optimum temperature for growth – 35 – 36 C. Essential to provide 5 – 10 % CO 2. Grow well on CHOCOLATE AGAR & MUELLUR – HINTON AGAR. Selective medium – THAYER – MARTIN medium (contains Vancomycin + Colistin + Nystatin which inhibit most of the contaminants).

8. CULTURAL CHARACTERS

9. BIOCHEMICAL REACTIONS Are oxidase positive . Are Catalase positive Rapid Carbohydrate Utilization test: Glucose Maltose N.gonorrhoeae + (Acid) -

10. ANTIGENIC PROPERTIES It is polyphosphate and not polysaccharide. Most evident on freshly isolated gonococci. Is loosely associated with cell surface. It inhibits phagocytosis. Are hair like structures extending from the surface Piliated organisms produce altered appearance of colonies on culture. Pilli enhance attachment of organism to host cells & prevent phagocytosis. Act as virulence factor . Are made up of pillin proteins. Pillin proteins are antigenically different in almost all strains. A single strain can produce several antigenically distinct pilli. 1. CAPSULE 2. PILLI

11. ANTIGENIC PROPERTIES There is absence of long O – antigenic side chains in the chemical structure of Lipooligosaccharide (LOS) which differentiates it from lipopolysaccharide of Gram – ve bacilli. Toxicity in gonococci is due to LOS. IgA1 protease is produced by gonococci. It splits and inactivates IgA which plays a major role in mucosal defense. Two other proteins H8 and Iron binding protein are also produced but their role in pathogenicity is unknown. 3.LIPOOLIGOSACCHARIDE (LOS) 4.OTHER PROTEINS

12. ANTIGENIC PROPERTIES 5. Proteins – The outer membrane antigens ( proteins ) are the porins. Protein I (por) – Forms pore on surface. Each strain expresses one type of protein I. It helps in serotyping of gonococci. Two variants of protein I – IA & IB. Any one strain carries either IA or IB but not both. 24 serovers of type IA & 32 serovers of type IB. Protein II (opa) – One part of protein II is in outer membrane & the rest is exposed on the surface of bacteria. It takes part in adhesion of bacterium and its attachment to host cell. Type II protein is present in those strains which form opaque colonies, so it is also c/a opacity associated protein . Protein III – is associated with protein I in the formation of pores on the cell surface & hence plays a role in the exchange of molecules across the outer membrane.

13. RESISTANCE & PLASMID Is very delicate organism Readily killed by drying, heat & antiseptics. Is a strict parasite & dies in 1 – 2 hrs in exudate outside the body. In culture, the coccus dies in 3 – 4 days but survives in slant culture at 35 C if kept under sterile paraffin oil. Cultures – preserved for years if frozen quickly & stored at – -- 70 C. Gonococci contains several cryptic plasmids. Two other transmissible plasmids contain genes that code for beta lactamase which causes resistance to penicillin. RESISTANCE PLASMID

14. PATHOGENESIS N.gonorrhoeae is Human pathogen. Chimpanzees can be infected artificially Women may remain Asymptomatic Gonorrhea infection is generally limited to superficial mucosal surfaces lined by columnar epithelium.

15. PATHOGENESIS THE ORGANS & TISSUES INVOLVED Cervix Urethra Rectum Pharynx Conjunctiva Vaginal epithelium – covered with squamous epithelium not infected However prepubertal vaginal epithelium is infected in young girls present with vulvovaginitis

16. PATHOGENESIS CONTD ………. Gonorrhea is a Venereal disease. The disease is acquired by sexual contact. In general incubation period is 2 -8 days. In men the disease starts as purulent discharge containing gonococci in large numbers. The disease spreads to prostate, seminal vesicle, and epididymis. In men chronic urethritis may lead to stricture formation. The infection may spread to the periurethral tissues, forming abscesses & multiple discharging sinuses ------------- “ Watercan perineum”.

17. PATHOGENESIS CONTD ………. CLINICAL PRESENTATION IN MALES – Majority of males present with acute urethritis with purulent discharge 5% of patients carry bacteria without distress Rectal and Pharyngeal infections are less often symptomatic

18. PATHOGENESIS CONTD ………. CLINICAL PRESENTATION IN FEMALES – Endocervix infection is the most common presentation in women Present with vaginal discharge and Dysuria Infection and abscess of Bartholin and Skene’s glands

19. PATHOGENESIS CONTD ………. – OTHER MANIFESTATIONS Peritonitis can spread. Fitz Hugh Curtis Syndrome --- Perihepatic inflammation. Disseminated gonococcus infection Painful Joints may lead to Arthritis Fever, Few septic lesions on the extremities Meningitis and Endocarditis

20. PATHOGENESIS CONTD ………. – GONORRHOEA CAN MANIFEST AS ORAL INFECTION - Changing sexual practices and oral sex predisposes the sex partners with involvement of oropharengeal regions

21. PATHOGENESIS CONTD ………. – DISSEMINATED INFECTION - Seen more commonly in women who may present with painful joints, fever, and few septic lesions or the extremities Rarely disseminated infections may present as endocarditis or meningitis

22. LABORATORY DIAGNOSIS 1. SPECIMENS - Discharge or urethral swab, Endocervical swab – - The meatus is cleaned with a gauze soaked in saline & a sample of the discharge collected with a platinum loop for culture, or directly on the slide for smears. - Cervical swabs are collected carefully, using speculum. - In chronic cases morning drop of secretion is examined. - Centrifuged deposits of urine also demonstrate gonococci.

23. LABORATORY DIAGNOSIS TRANSPORTATION OF SPECIMENS - It is preferred in culture, specimens should be inoculated in prewarmed plates,immeditely on collection If not possible specimen should be collected on charcoal impregnated swabs and sent to laboratory in Stuart’s transport medium.

24. LABORATORY DIAGNOSIS - MICROSCOPY – GRAM STAINING Gram’s method of staining is sensitive in 95% of infections. The demonstration of intracellular Gram negative diplococci in stained smears provides a presumptive evidence of gonorrhea in men. Diagnosis of gonorrhoea by smear exam. is unreliable in females. Specimens are inoculated onto culture plates freshly prepared. Delay in inoculation of specimens on culture media reduces the rate of isolation.

25. LABORATORY DIAGNOSIS - CULTURE - Gonococci are aerobic and may grow aerobically. It is essential to provide 5 – 10% CO 2. They grow well on Chocolate agar and Mueller – Hinton agar. The colonies are small, convex, translucent, and slightly umbonate with finely granular surface and lobate margins.

26. LABORATORY DIAGNOSIS - SELECTIVE CULTURE MEDIUM - The selective medium is Thayer – Martin medium containing Vancomycin, colistin, and Nystatin , effectively inhibits most contaminants including non pathogenic Neisseria

27. LABORATORY DIAGNOSIS - SEROLOGY - Complement fixation test Precipitation Reaction Passive agglutination Using whole cell lysate, pilus protein Immunofluorescence & lipopolysaccharide Ags. Radioimmunoassay Coagglutination - Biochemical Tests – Catalase Test - +ve, Oxidase Test - +ve Ferments glucose with acid production. Maltose not fermented.

28. LABORATORY DIAGNOSIS - MOLECULAR METHODS - DNA probes can be used for confirmation, and also used for the detection of Gonococci in urethral and cervical specimens. PCR methods are available in specialized laboratories

29. TREATMENT - PENICILLIN - Was a popularly used antibiotic. However resistance has reduced its utility. For a long time the drug resistance has overcome with increased dosage. Complete resistance to penicillin has made the drug obsolete in several parts of the world.

30. TREATMENT - PENICILLIN - GENES CODE RESISTANCE The penicillin resistant strains possess the gene coding for TEM-type ß-lactamase commonly found in Escherichia coli.

31. TREATMENT - OTHER DRUGS USED - Ceftriaxone Cefixime Flouroquinolones Ciprofloxin Tetracycline Co- Amoxi - clav Spectinomycin In Disseminated Gonococcal disease and any complicated infection treatment for 7-10 days is necessary

32. CONTROLING GONORRHOEA The key control measures in gonorrhea are - 1 Rapid diagnosis 2 Use of effective antibiotics 3 Tracing, examination and treatment of contacts. 4 Inappropriate self medication has contributed to widespread antimicrobial resistance.

33. 33 THANKS