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Neisseria

Neisseria
gonorrhoeae
gonorrhoeae
and
and
Gonorrhea
Gonorrhea
Sridhar Rao P.N Sridhar Rao P.N
Assistant Professor Assistant Professor
Dept. of microbiology Dept. of microbiology
JJMMC, Davangere JJMMC, Davangere
www.microrao.com
Etiology
Etiology
Neisseria gonorrhoeae is an oxidase positive, gram-
negative, non-motile, and non-spore forming
diplococci, 0.6 to 1.0 m in diameter, which grows in
adjacent pairs
Often observed as kidney/bean shaped diplococci
inside polymorphonuclear leucocytes or even
extracellularly
The outer membrane is composed of proteins,
phospholipids, and lipopolysaccharide (LPS).
The highly branched basal oligosaccharide structure
and the absence of repeating O-antigen subunits
makes them different from enteric LPS. The
gonococcal LPS is referred to as lipooligosaccharide
(LOS).
Gonococci characteristically release outer membrane
fragments (blebs) during growth. These blebs contain
LOS and may have a role in pathogenesis.
Colony morphology
Colony morphology
Five colonial types have been described, which can be
identified using steromicroscope.Freshly isolated
strains consists of types 1 and 2.
Type 1 is small, raised, slightly viscid, dewdrop colony
whereas type 2 is small, raised and friable. Types 1
and 2 reflect incident light and appear glistening.
During non-selective transfer, type 1 and 2 revert to
types 3 to 5, which are larger, slightly convex and do
not reflect incident light.The type 1 and 2 are piliated,
which are denoted P+ while Type 3, 4 and 5 are
nonpiliated, and are denoted P-
Nonpiliated colonies can revert back to being P+,
these reversions happen by antigenic and phase
variation at very high frequencies.
Colonies can also be opaque or transparent, the
opaque colonies contain Opa and the transparent do
not.
Pathogenesis
Pathogenesis
Infection occurs following sexual contact.
The disease is of high infectivity and the risk of
infection is much greater for women
Anal infection in men is always the result of rectal
sex with an infected partner.
Gonococcal pharyngitis always results from the
practice of fellatio.
Gonorrhea occurring in the vagina or rectum of
prepubertal children is usually transmitted from
adults through sexual abuse or, rarely, by fomites.
Mother to child transmission also occurs as the child
passes through the birth canal during delivery
causing eye infections
Medical Microbiology, 4th Edition,
Samuel Baron, MD
In adults, the mucous membranes that are lined by
columnar, nonciliated epithelial cells are vulnerable to
gonococcal infection. Attachment to ciliated cells does
not occur.
Squamous epithelium, which lines the adult vagina, is
not susceptible to infection by the gonococcus.
However, the prepubertal vaginal epithelium, which
has not been keratinized under the influence of
estrogen, may be infected. Hence, gonorrhea in
young girls may present as vulvovaginitis.
They usually affect epithelial cells of the urethra in
males and of the endocervix in females
Gonococci use their pili, protein I (porin protein)1
and protein II (opacity protein) and other surface
proteins to adhere to the nonciliated epithelial cells.
Opa proteins are expressed by certain gonococci and
are used advantageously to adhere to many cells that
contain the host cell receptor CD66. Nonspecific
factors, such as surface charge and hydrophobicity can
play a role
Attachment is followed by mucosal damage, which is
manifestedas loss of ciliary activity and sloughing of
ciliated cells.
This is thought to be mediated by peptidoglycan and
lipopolysaccharide
After adhesion, the gonococci are pinocytosed (a
process called parasite-directed endocytosis) by the
epithelial cells inside a vacuole, which is transported,
from the mucosal surface to the bottom of the cell
where the gonococci are released by exocytosis into
the subepithelial tissue.
Gonococci multiply and divide while inside of the
epithelial cell creating many bacteria cells to be
released into the subepithelial tissue.
After invasion of the sub-epithelial tissues, the
gonococci elicit an inflammatory response, which is
responsible for typical symptoms of gonorrhea.
Mucosal invasion may lead to bacteremia and
subsequent dissemination.
Strains with requirements for arginine, hypoxanthine,
and uracil (AHU strains) are predominant among
isolates from disseminated gonococcal infections
These isolates possess protein I in outer membrane
protein, are resistant to normal bactericidal action of
serum, are more resistant to penicillins, and are
resistant to phagocytosis
Serum resistance may be due to protein I and
difference in their structure of polysaccharide
Antigenic variation of the opacity protein and pili
occurs during infection.
This antigenic variation results in persistence of
infection and colonization of urethra, cervix and
rectum
Variations in Opa has been associated with attachment
to PMNLs, epithelial cells and resistance to
antimicrobials and serum killing
Gonococci is also demonstrates antigenic variation in
lipopolysaccharides.
All gonococci also produce IgA1 proteases cleave IgA1
and release Fab and Fc fragments
lipooligosaccharide (LOS) is known to induce
production of tumor necrosis factor (TNF) that causes
cell damage and is also involved with the drug
resistance of N. gonorrhoeae.
Ability to extract iron is another virulence factor.
Women are frequently asymptomatic carriers of the
organism for weeks or months and often are identified
when sexual contacts are traced.
In men, the incubation period is from 2 to 14 days.
Onset is usually marked by mild discomfort in the
urethra, followed a few hours later by dysuria and a
purulent discharge. Frequency and urgency of
micturition develop as the disease spreads to the
posterior urethra.
The most common symptom of uncomplicated
gonorrhea is a discharge that may range from a scanty,
clear, or cloudy fluid to one that is copious and
purulent.
Men with asymptomatic urethritis are an important
reservoir for transmission.
Gonorrhea
Gonorrhea
In women, symptoms usually begin within 7 to 21
days after infection.
Though symptoms generally are mild, onset is
sometimes severe, with dysuria, frequency, and
vaginal discharge. The cervix and deeper
reproductive organs are the sites most frequently
infected, followed by the urethra, rectum, Skene's
ducts, and Bartholin's glands.
Endocervical infection is the most common form of
uncomplicated gonorrhea in women. Such infections
are usually characterized by vaginal discharge and
sometimes by dysuria (because of coexistent
urethritis).
About 50 percent of women with cervical infections
are asymptomatic. Local complications include
abscesses in Bartholin's and Skene's glands.
In women or homosexual men, rectal gonorrhea is
common. Women are usually asymptomatic, but
perianal discomfort and a rectal discharge may occur.
Severe rectal infection is more common in homosexual
men.
Rectal infections with N. gonorrhoeae occur in about
one-third of women with cervical infection. They most
often result from autoinoculation with cervical
discharge and are rarely symptomatic.
Gonococcal pharyngitis from orogenital contact is
usually asymptomatic, but some patients complain of
a sore throat and discomfort on swallowing; the
pharynx and tonsillar area may be red, exudative, and
occasionally edematous
In female infants and prepubertal girls, irritation,
erythema, and edema of the vulva with a purulent
vaginal discharge may be accompanied by proctitis.
Ocular infections (ophthalmia neonatorum) occur
most commonly in newborns who are exposed to
infected secretions in the birth canal.
Keratoconjunctivitis is occasionally seen in adults as a
result of autoinoculation. Asymptomatic infections of
the pharynx, urethra, or cervix often serve as focal
sources for bacteremia.
Disseminated gonococcal infections result from
gonococcal bacteremia. The most common form of
disseminated gonococcal infection is characterized by
fever, chills, skin lesions, and arthralgias .
Skin lesions may be macular, pustular, centrally
necrotic, or hemorrhagic.
Rarely, disseminated gonococcal infection causes
endocarditis or meningitis.
Gonococci may ascend from the endocervical
canal through the endometrium to the fallopian
tubes and ultimately to the pelvic peritoneum,
resulting in endometritis, salpingitis, and finally,
peritonitis.
Women usually present with pelvic and
abdominal pain, fever, chills, and cervical motion
tenderness. This complex of signs and symptoms
is referred to as pelvic inflammatory disease
(PID).
Complications of pelvic inflammatory disease
include tubo-ovarian abscesses, pelvic peritonitis,
or Fitz-Hugh and Curtis syndrome
Laboratory diagnosis
Laboratory diagnosis
Specimen collection depends on the site of infection.
Patient should not urinate 2 hours prior to specimen
collection
Urethral specimens may be obtained from men by
inserting a thin calcium alginate, dacron or rayon
swab or an inoculating loop 2-3 cms inside the
urethral orifice.
In homosexual men, pharyngeal, rectal and urethral
specimen must be collected
Rectal swab is collected by inserting cotton swab 1
inch inside the anal canal and moving the swab side to
side for 30 seconds. Rectal mucosa can also be taken
by a anoscope.
Endocervical swabs are collected using a cotton swab
after the cervix is cleaned with a vaginal speculum
and is rotated in circular motion.
Other specimen
Other specimen
Blood for blood cultures should be drawn from
patients suspected of disseminated infection
Biopsies of skin lesion and synovial fluid if infected
Specimens may also be obtained from the urethra
and from Bartholin's and Skene's glands of infected
women.
Swabs of conjunctival exudates from infected infants
eye
Cerebrospinal fluid should be collected if signs or
symptoms of meningitis are present.
Appropriate specimen must also be collected from
the patients partner
Ideally, specimen should be plated into culture media
and incubated immediately.
In case of delay transport medium (Stuarts or Amies)
may be used, these tubes should be airtight, almost
filled with medium and inoculation should be done
with minimum exposure to air. Holding medium
should not be used for transport beyond 6 hours
If cotton swabs are used, transport medium must
contain charcoal to neutralise the toxic effects of
cotton fibers
For longer transport, Transgrow medium (containing
modified Thayer-Martin agar and CO
2
in flat bottles)
can be used.
Jembec system is useful in case of delays exceeding
18-24 hours. The system contains flat dish with
culture medium and a well for tablet that generates
CO
2
.
Microscopy
Microscopy
Gram smear on urethral exudate is reliable upto 95%
in symptomatic men. Direct smears of rectal swabs
are not reliable, however material collected from
anoscope can be useful.
Gram stain is not useful for the diagnosis of
pharyngeal infection because the oropharynx may be
colonized by other Neisseria species Sensitivity of
smears on endocervical swabs in symptomatic women
is 60%.
Smears are stained with Gram stain. Modifications
include use of Sandiford as counterstain
Gonococci can also be viewed using
immunofluorescence.
After inoculating into the medium, the tablet is placed
in the well and sealed in a pouch and incubated for 18-
24 hours before shipping to the laboratory. GonoPack
is another commercial alternative.
Male Urethral Smear
Positive: 1 PMN with intracellular Gram-negative
diplococci of typical morphology. Extracellular Gram-
negative diplococci may also be present, and
numerous PMNs are usually present.
Negative: No intracellular Gram-negative diplococci.
Cervical Smear
Positive: 1 PMN clearly containing Gram-negative
diplococci of typical morphology. Numerous PMNLs,
extracellular Gram-negative diplococci, Gram-
negative rods and Gram-positive rods may be seen.
Not diagnostic: No intracellular Gram-negative
diplococci or only extracellular Gram-negative
diplococci found. PMNs may be present.
Culture
Culture
Specimen collected for culture must not be
refrigerated
Urethral, rectal and cervical swabs must be cultured
for specificity
Identification of the gonococcus by culture in genital
exudate should be attempted for all women and for
men with negative or equivocal urethral Gram stains.
Exudates must be inoculated onto a suitable medium
(eg, modified Thayer-Martin medium, Martin-Lewis, or
NYC medium) and incubated at 35 to 36 C for 48 h in
an atmosphere containing 3 to 10% CO
2
(candle jar or
CO
2
incubator).
Other culture media include GC-lect agar and modified
NYC agar. Strains susceptible to Vancomycin may be
grown on Chocolate agar
If plates cannot be incubated immediately, they can
be held safely for several hours at room temperature
in candle extinction jars prior to incubation.
Identification
Identification
Oxidase test must be performed on the colonies
The Superoxol test (catalase test using 30% H
2
0
2
) can
be used to differentiate Neisseria gonorrhoeae from
other Neisseria species.
Tests for confirmation of N. gonorrhoeae include
carbohydrate utilization, coagglutination reactions,
and chromogenic substrate tests.
Carbohydrate utilization tests include Rapid
fermentation test, semisolid Cystine-Trypticase agar
(CTA) technique and the BACTEC Neisseria
differentiation kit.
The RFT identifies organisms after 1 to 4 hr of
incubation, BACTEC radiometric method identifies
organisms after 3 hr of incubation, and the CTA
technique identifies organisms after 24 to 48 hours of
incubation.
Staphylococcal coagglutination technique developed
for the identification of N. gonorrhoeae identifies it in
2 min.
Prolyliminopeptidase (PIP) reaction test relies on
demonstration of activity of this enzyme.
Development of yellow color after 2 hour incubation
period of the gonococcus with the substrate L-proline
-naphthylamide hydrochloride is positive.
Gonococcal strains can be characterized according to
their nutritional requirements (auxotyping).
A panel of monoclonal antibodies specific for epitopes
on protein I have also been used to type strains.
A combined auxotype-serovar classification provides
greater resolution among gonococcal isolates and is
useful in epidemiologic investigations.
Other techniques
Other techniques
Commercially systems such as Vitek, Gonochek II,
RapID INH panel are also available.
Antigen detection in urine sediments by enzyme
immunoassay
Detection of gonococcal genes by DNA hybridization
tests, PCR or LCR
Nucleic acid amplification tests are available for the
detection of N. gonorrhoeae in urethral swab
specimens obtained from males, endocervical swabs,
and urine specimens obtained from men and women.
NAATs can be used on eye and vaginal secretions, but
are not recommended for rectal and pharyngeal
specimens
Indirect fluorescent antibody technique for the
serological diagnosis of gonorrhea.
Treatment
Treatment
The emergence of drug-resistant gonococci has
limited the usefulness of previously recommended
penicillin-, ampicillin-, and tetracycline-based
regimens.
A single dose of ceftriaxone for gonococci plus either
doxycycline or azithromycin for chlamydia is
recommended as initial therapy for urethral,
endocervical, pharyngeal, and rectal infections.
Alternative to ceftriaxone are a single dose of either
spectinomycin, ciprofloxacin, ofloxacin or cefixime.
All regimens should be accompanied by azithromycin
or doxycycline to treat possible chlamydial co-
infection, except in pregnant women.
Drug resistance
Drug resistance
Resistance to penicillins may be either plasmid or
chromosome mediated
Plasmid mediated resistance is mediated by
production of beta-lactamase (PPNG)
Chromosomally mediated resistant N. gonorrheae
(CMRNG) involve alteration of the permeability of the
OMP or decreased affinity of PBPs
Increasing numbers of gonococci with decreased
susceptibility to third generation cephalosporins are
found in several countries
There are infrequent instances of spectinomycin
resistance
Quinolone-resistant N. gonorrhoeae (QRNG) have
been reported in some countries