The neisseriae CLASSIFICATION Kingdom Phylum Bacteria Proteobacteriacea Class

The neisseriae

CLASSIFICATION Kingdom Phylum Bacteria Proteobacteriacea Class Betaproteobacteria Order Neisseriales Family Neisseriaceae Genus Neisseria Kingella, ikenella Species (Neisseria) Gonorrhoeae Meningitidis

COMMENSAL NEISSERIAE • • • N. lactamica N. pharyngis N. cinerea N. flavescens N. mucosa

• The Neisseriaceae are gram negative cocci which commonly occur in pairs. • The inhabit the mucus membranes of humans (nasopharynx)

Neisseria meningitidis (meningococcus) • It is the causative agent of meningococcal meningitis. • A gram negative diplococcus with adjacent sides flattened. • Fresh isolates are capsulated & are having pilli.

SEROGROUPS AND SEROTYPES Serogroups On the basis of specificity of capsular polysaccharide antigens divided into 13 serogroups. • A, B, C, D, X, Y, Z, W -135, 29 -E, H, I, K and L. Serotypes Based on the outer membrane protein serogroups are further divided into serotypes. About 20 serotypes have been identified.

Serogroups A, B, C, X, Y, W 135 commonly associated with meningococcal disease. • Group A: epidemics • Group C: localised outbreaks • Group B: both epidemics and outbreaks

Pathogenesis of meningococcal meningitis Natural habitat and reservoir • Human nasopharynx • Urogenital tract • Anal canal Nasopharyngeal carriers 5 -10% adults asymptomatic carriers Modes of infection Direct contact Respiratory droplets from the nose and throat of infected people

Source of infection a) Nasopharyngeal carriers b) Patients Note -> ü Carriage rate between epidemics (5 – 30%) ü During epidemics (70 – 80%)

Bacterial virulence factors Capsular polysaccharide, Antiphagocytic, Antigenic, Outer membrane proteins Help in adhesion to host epithelia. Lipopolysaccharide Responsible for the toxic effects Cell membrane proteins Fimbrae (common pili)- Adhesion, Invasion

Host predisposing factors Invasive infection is favored by certain predisposing factors -> ü Absence of Bactericidal Abs (Ig. M, Ig. G) ü Complement deficiency (C 5, C 6, C 7, C 8) ü Inhibition of serum bactericidal action by a blocking Ig. A Ab

SEQUENCE OF EVENTS Inhalation of contaminated droplets Adherence of organism to nasopharyngeal mucosa Local invasion Blood stream Along perineural sheath of olfactory spread from nasopharynx to nerve, cribriform plate to meninges subarachnoid space In meninges, organsims are internalised into phagocytic cells They replicate and migrate to subepithelial spaces Incubation period : 3 -4 days

Infection may take either of 3 courses -> Meningitis Fulminant meningococcemia Chronic infection

Meningitis ü Most common complication of meningococcemia ü Meninges are acutely inflamed with thrombosis of blood vessels & exudation of PMNLS ü Sudden onset of Ø Intense headache, Ø vomiting, Ø stiff neck –> progresses to coma within a few hours.

Fulminant meningococcemia An acute condition characterized by ü High fever ü Hemorrhaging of internal organs ü Hemorrhagic skin rashes ü DIC with circulatory collapse ü Known as the “Waterhouse – Freiderichsen syndrome”

Chronic type of infection • Rare occurrence • Recurrent episodes of –> - fever - arthritis - maculopapular rash • Condition may last for weeks / months

Lab diagnosis • As meningitis is caused by several organisms, it is important to identify the etiology. Specimen CSF – Sample should be processed immediately. If delay in processing, should be incubated at 37 o. C not refrigerated. Should be examined by -> Smear Culture Biochemical determination

B. Blood - for culture C. Nasopharyngeal swab Ø For determination of carriage Ø Should be transported in Stuart’s transport medium • • Skin lesions Joint fluid Tracheal aspirate Petechial lesions

Processing A. CSF -> Should be divided into 3 portions 1 st portion -> Centrifuged Deposit is used for smear preparation • Gram stained smear: - gram negative diplococci within and outside PMNLs. • Supernatant: - used for capsular Ag detection using serological tests

2 nd portion -> Inoculated onto Chocolate / Blood agar Incubated at 37 o. C for up to 48 hrs at 5% CO 2 Ø Colonies growing on these media are identified as meningococci by -> a) Colony characters Bluish grey, Glistening, Convex Smooth, Translucent colonies.

b) Morphology – Gram negative diplococci with flat sides adjacent. c) Biochemical reactions - ü Oxidase test - Positive ü Glucose utilization test – Positive ü Maltose utilization test – Positive ü Sucrose utilization test – Negative d) Serogrouping / serotyping may be done using specific antisera

3 rd Portion of CSF -> Cell count, Glucose & protein determination is done to determine the type of meningitis Diagnosis Cell type, Cell/µL Glucose Protein Normal Lymphocytes 0 -5 45 – 85 mg/d. L 15 – 45 mg/d. L Pyogenic meningitis PMNLS 200 – 20000 Low (< 45 mg/d. L) High (> 50 mg/d. L) Aseptic meningitis Mostly lymphocytes, 100 – 1000 Normal Moderately high (> 50 mg/d. L)

B. Blood -> ü Blood is immediately inoculated onto blood culture media such as BHI broth, incubated up to 7 days. ü S/C is done onto chocolate agar & colonies are identified as previously described. C. Nasopharyngeal swabs -> Inoculated onto selective media containing antibiotics such as vancomycin, colistin, amphotericin B Ø Modified Thayer – Martin medium Ø New York City medium

Detection of antigen • For Detection of Meningiococcal DNA -Polymerase Chain Reaction (PCR) • For detection of soluble polysaccharide antigen - Counter current immunoelectrophoresis (CIEP) - Latex agglutination test

Treatment • Penicillin G is the drug of choice • In people allergic to penicillin, Ceftriaxone or Cefotaxime may be used

Prevention & Control Vaccine -> Capsular polysaccharide of serotypes A and C : for infants below 2 years A quadrivalent vaacine constituted by polysaccharides of serotypes A, C, Y and W-135 : for children and adults. Conjugate vaccine: • polysaccharide antigen is conjugated to diptheria • toxoid

Clinical cases -> should be isolated Carriers / contacts -> Ø Oral rifampin (600 mg bd for 2 days) Ø Ciprofloxacin (500 mg, single dose)

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Neisseria gonorrhoeae (Gonococcus)

• Exclusively a human disease. • First described by Neisser in 1879 in gonorrheal pus. • In one of the top 5 STD’s. • Almost always, sexually transmitted except ophthalmia neonatorum. • Does not survive more than 1 - 2 hours on fomities.

• Infective dose is 100 -1000. • Chance of acquiring infection after single exposure -> In men (20 – 30%). In women (>30%). • Infection is asymptomatic in 10% males and 50% of females.

GONOCOCCUS – • Gram negative diplococci usually found with in the polymorphs. • Oval/spherical kidney (bean) shaped. • Arranged in pairs with the concave sides facing each other. • Possess pili on the surface. • Fastidious organisms do not grow on ordinary culture media. • Aerobic but may grow anaerobically also.

• The optimum temperature for growth is 35 -36°C & optimum p. H is 7. 2 to 7. 6. • It is essential to provide 5 -10% CO 2. Gonococcus iside the polymorhs

Bacterial Virulence factors PEPTIDOGLYCAN PILUS üAttachment , resistance against phagocytosis. üSwitch Ags to evade immune responses Por PROTEINS Help in intracellular survival by preventing phagosome-lysosome fusion Opa PROTEINS adhesion to host cell receptors LOS Toxicity ü Evading immune recognition Inhibits cidal activity of serum IGA PROTEASE splits & inactivates Ig. A 1 provides protection to mucosal surfaces. CAPSULE resistance again phagocytosis

Pathogenesis & Clinical findings 1. Source of infection -> Infected patient, with clinical / subclinical infection 2. Mode of infection -> By sexual contact (except ‘ophthalmia neonatorum’ wherin infection occurs while passing through the infected birth canal) 3. Incubation period -> 1 – 14 days

Sequence of events & clinical manifestations Gonococci attack the mucus membranes genito-urinary tract, eye, rectum & throat Introduction onto mucosal surfaces Gonococci adhere strongly to the mucosal surface Phagocytosed by the epithelial cells Transported in vacuoles to the base of the epithelial cells Released into the sub-epithelial tissues Elicit an intense inflammatory reaction

Primary site of infection Ø In males -> Urethra Ø In females -> Endocervix Ø In both sexes -> Pharynx & ano-rectal region

In men The disease starts as an acute urethritis with a mucopurulent discharge Extends to the prostate, seminal vesicles & epididymis In some it may become chronic urethritis leading to stricture formation The infection may spread to the periurethral tissues, causing abscesses & multiple discharging sinuses (Watercan perineum)

In women The initial infection is urethritis & cervicitis but vaginitis does not occur in adult female (vulvovaginitis can occur in prepubertal girls) The infection may extend to Bartholin’s glands, endometrium & fallopian tubes causing Pelvic Inflammatory Disease (PID) Rarely peritonitis may develop with perihepatic inflammation (Fitz-Hugh-Curtis syndrome)

In both sexes Anorectal infection In men – due to rectal intercourse by homosexuals. In women – usually due to perineal contamination with the mucopurulent discharge. Bacteraemia which may lead to metastatic infection such as arthritis, endocarditis, meningitis, pyemia & skin rashes. Pharyngeal infection Due to orogenital sex, usually, asymptomatic.

Ophthalmia neonatorum Ø Infection of the eye of the new born. Ø Infection is acquired during passage through the infected birth canal. Ø Initial conjunctivitis, if untreated, will lead to blindness.

Complications seen in gonococcal infections • More frequent in women than men • Gonococcal bacteremia leads to Disseminated gonococcal infection (DGI) Symptoms of DGI ü Hemorrhagic rashes on hands, forearms, legs & feet. ü Tenosynovitis & suppurative arthritis of knees, ankles & wrists. ü Infertility. ü Endocarditis & Meningitis are rare complications

Lab diagnosis A. Specimen collected 1. Pus & secretions for smears & culture collected from Ø Urethra Ø Cervix Ø Rectum, Ø Throat Ø Conjunctiva 2. Synovial fluid -> for smears & culture. 3. Urine -> for smears & culture. 4. Blood -> collected for blood culture in case of systemic illness.

ü Specimen collected in Acute cases a) In men -> • The meatus is cleaned with saline and urethral exudate is collected with the help of a platinum loop or charcoal impregnated swab. • For culture, sample is immediately, directly inoculated onto appropriate culture media. • If there is delay in processing, transport media such as Stuart’s or Amie’s media should be used.

b) In women -> Ø urethral exudate & cervical samples (collected using a speculum) should be taken. ü Specimen in chronic cases Ø Little or no urethral discharge Ø Urethral exudate collected after prostatic massage Ø Morning drop of secretion Ø Centrifuged deposits of urine

B. Microscopy -> 1. Gram stained smear of exudates –> will show numerous pus cells, gram negative diplococci both intracellular and outside of pus cells

• Diagnostic 90% sensitive, 99% specific. ü In acute urethral cases from men ü In conjunctival exudate from new borns • Less reliable 50% sensitive, 95% specific. due to presence of commensal neisseriae ü In endocervical samples from females ü Pharyngeal & anorectal samples

Culture Medias used to grow the Gonococcus Ø For urethral samples from acute cases –> Chocolate / Blood agar. Modified New York City medium. • For all other samples -> Modified Thayer – Martin medium containing antibiotics (Vancomycin, Colistin & Nystatin) is used.

• • • Small Round Translucent Convex or slightly umbonate finely granular surface lobate margins.

Ø Identification of isolate is done by ü Gram stained smear – gram negative diplococci ü Oxidase test – Positive ü Glucose utilization test – Positive ü Maltose utilization test – Negative ü Sucrose utilization test – Negative G N diplococci

C. Serology -> • Complement fixation test, • Precipitation, • Passive agglutination, • Immunofluorescence, • Radioimmunoassay. (uses whole-cell lysate, pilus protein and lipopolysaccharide antigen) D. Nucleic acid amplification test -> Directly detects gonococcal DNA in samples

Treatment Ø Penicillinase producing N. gonorrheae (PPNG) have emerged which show high level of resistance to penicillin Ø Tetracycline & Fluoroquinolone resistance has also been demonstrated

ü Acute cases are treated with -> Procaine Penicillin G (Single dose, IM, 2. 4 – 4. 8 Million Units) ü Ceftriaxone (single dose, IM, 250 mg) ü Doxycycline (100 mg, bd, for 7 days, for concomitant Chlamydial infection)

Prevention & Control Ø No vaccines available due to heterogeneity of strains ü Avoiding multiple sexual partners ü Early diagnosis & treatment of cases ü Education ü Screening population at high risk ü Mechanical prophylaxis (Condoms) provide partial protection

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