INTRODUCTION
 CHLAMYDIAE: Obligate intracellular bacteria
 Family- CHLAMYDIACEAE

 Classification of Family Chlamydiaceae (2 genera)
Genus Chlamydia
Genus Chlamydophila
C.trachomatis
C.pneumoniae
C.psittaci
3 species causing human disease:
 C.trachomatis
 C.psittaci
 C.pneumoniae
C.trachomatis
Trachoma
Lymphogranuloma Venerum
C.psittaci
C.pneumoniae
Psittacosis
Atypical pneumonia
FEATURES
1. Small obligate intra cellular Gram negative bacteria:
poorly stained by Gram’s stain
2. Possess both RNA & DNA, ribosomes, cell wall similar
to Gram negative bacteria.
3. Absence of peptidoglycan is the only difference b/w
other Gram -ve bacteria.
4. They lack ability to generate their own ATP. So use
host ATP.
5. Divide, multiply by binary fission



Readily stained by GIEMSA, CASTANEDA, GIMENEZ,
MACHIAVELLO methods.
-Castaneda - blue colour
-Gimenez and Machiavello method- red in color
-Giemsa: method of choice for staining inclusion bodies in
cell culture- they are basophilic

Mature C.trachomatis possess inclusion of Glycogen
matrix- so stained by iodine- coppery brown color

C,psittaci has no glycogen, so not stained by iodine
Demonstrated by direct immuno-fluorescence
They multiply in the cytoplasm of the host cell forming
micro colonies or inclusion bodies which drape
around the nucleus like a cloak- “Chlamys” means
mantle.
Antigenic structure:
Chlamydiae possess the following antigens:
 Genus specific Ag: Lipopolysaccharide (LPS): Imp in
pathogenesis by induction of TNF-α: leading to scarring
and fibrosis.

Species specific protein Ag:

Serovar specific Ags: Major Outer Membrane Protein
(MOMP) used in micro-immunoflourescence

They infect wide spectrum of vertebrate hosts- birds,
mammals and humans.

Susceptible to wide range of antibiotics- tetracycline,
erythromycin, macrolides, rifampicin.

C.trachomatis also sensitive to sulfonamides whereas
C.psittaci and C.pneumoniae are resistant
DEVELOPMENT / LIFE CYCLE
 Chlamydiae exist in 2 distinct forms-
1. EB- Elementary Body 2. RB- Reticulate Body
ELEMENTARY BODY(EB):

Extra-cellular infectious particle

Small (250-350nm diameter)

Spherical in case of C.trachomatis and C.psittaci. Pear
shaped in C.pneumoniae

Has irregular electron dense nucleoli

Capable of extra cellular survival
 RETICULATE BODIES(RB)-
1. Intra cellular
2. Metabolically active
3. Divides by binary fision to form EBs.
4. 800-1200nm in diameter
5. Cell wall lacks disulfide cross linking
ATTACHEMENT:
 Infection is initiated by the attachment of infectious EB to
susceptible host cells
Eg: Biovars- Trachoma- Squamous, columnar cell
C.trachomatis- LGV- lymphoid cell
C.psittaci- wide range of cells
INTRACELLULAR SURVIVAL: Organism enters host cell
within a vesicle. Chlamydiae dependant modification of
endocyte membrane prevents the lysosomal fusion and
thus prevent degradation.

Conversion of EB to RB: 9 hours after infection - EB
within the vesicle loses its dense DNA core, cell wall
becomes less rigid due to breaking of disulfide bonds,
increases in size and differentiates into RB.

18 hours after infection- the vesicle is enlarged, RB divides
by binary fission, yields pleomorphic organisms and
genus specific chlamydial antigens become associated with
the host cell surface.

By 24 hrs: condensation of DNA within the RB, disulfide
bonds are formed in the outer membrane protein and new
EBs develop within the vesicle.

Formation of chlamydial micro colonies within the
vesicle is termed an INCLUSION BODY (IB).
It is typically peri-nuclear and may have 100-500 EBs.

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By 40-70 hrs: Infectious EBs are released from the cell by
rupture and these may infect new cells.
CHLAMYDIA TRACHOMATIS:
 Human pathogen causing: ocular, genital & neonatal
infections
 C.trachomatis possess 3 biovars,
a) Causing trachoma & inclusion conjunctivitis (TRIC)
b) Those causing LGV
 Based on antigenic structure of MOMP 18 serovars of
C.trachomatis are identified;
 PATHOGENICITY – Chlamydiae produce infection of
eye, male and female genital tract infection and
respiratory tract infections.
C.trachomatis serovars A, B, Ba & C
 Trachoma – Communicable chronic keratoconjunctivitis
 Transmitted through direct contact with eye discharge
from infected patients (fingers, flies fomites)
 Common in young children
 Acute infection: Follicles, papillary hyperplasia, pannus
formation and in late stages cicatrization, blindness.
 Causes blindness in trachoma belt – North Africa to
South east Asia, Middle east, North India
Inclusion – conjunctivitisC.trachomatis biovar TRIC serovars D-K.
 Natural habitat: genital tract of both sexes.
 Sexually active young people, spread from genitalia to
eye.
 Onset is acute, intense hyperemia, muco purulent
discharge & follicular hyperplasia.
 Unlike trachoma lesions most pronounced in the lower
than upper lid.
3. Opthalmia neonatrum [Inclusion blenorrhoea]
 Neonatal form of inclusion conjunctivitis.
 Infection acquired through birth canal.
 5-12% pregnant woman have chlamydial infection of
cervix, 50% infants born to such mothers will have
conjunctivitis.
 5-12 days after birth – neonate has swelling of eye lids,
hyperemia and a purulent infiltration of conjunctiva.
 A proportion of untreated neonates develop
pneumonia.
GENITAL INFECTIONS:
 Serovar D-K – cause 30% cases of NGU
 Other organisms of NGU are – Ureaplasma urealyticum,
Mycoplasma genitalium, M.hominis, Bacteroides
urealyticus, CMV, T.vaginalis.
 C.trachomatis also responsible for 50% cases of
epidydimitis in men under 35 years of age and 15% men
above 35 years.
 Females – C.trachomatis serovars D-K cause urethritis
mucopurulent cervicitis, vaginitis and vaginal discharge,
endometritis, salphingitis, infertility, perihepatitis and
periappendicitis
 Endometritis and salphingitis singly or together is called
PID [Pelvic Inflammatory Disease]
 After 1-2 months – regional lymph node [inguinal in male
and intrapelvic and pararectal in females] become enlarged
and tender and may break open with sinuses. These enlarged
lymph node are called BUBOS.
RESPIRATORY TRACT INFECTION:
 TW 183 and AR 39 isolated in 1965 from eye of child with
trachoma in Taiwan. Same strains also isolated from
pharyngitis throat.
 These two organism were called TWAR.
 But now classified as C.pneumonia.
 It appears as the third most common cause of pneumonia
after S. pneumoniae & H.influenzae.
Chlamydophila psittaci:
 It also causes acute lower respiratory infection in man.
 Psittacosis is disease of birds belonging to psittacine
family-parrots, transmissible to man (Zoonotic disease).
 Infection in birds exists as asymptomatic, latent infection
which can flare up following stress to birds like caging,
overcrowding.
 There will be diarrhea, mucopurulent respiratory
discharge and emaciation.
 Fecal discharge and nasal discharge of these birds will
contain many organisms so a source of infection to other
birds and humans.
 Man gets infection by inhalation of dried feces of birds.
 Incubation period 1-2 weeks.
 Clinical disease – mild influenza like syndrome. –
fever, general malaise, anorexia, rigors, sore throat and
photophobia.
 Some will have severe disease like pneumonia,
septicemia, meningo-encephalitis, pericarditis,
myocarditis, endocarditis, arthritis or a typhoid like
syndrome having enlarged liver, spleen and a rash.
LABORATORY DIAGNOSIS:
 Collection of specimens – Ocular, urethral, vaginal, cervical
specimen are best collected by scraping the mucosa.
 Depending on the site of involvement: blood, respiratory
secretions, sputum can be collected.
 In LGV pus from bubo to be collected.
PROCESSING OF SPECIMENDirect detection of chlamydial antigens.
1. Light microscopy: infection of conjunctiva, urethra can
be diagnosed – demonstration of typical inclusion
bodies surrounding the nucleus [HP bodies]
1.
Stain by Giemsa, Castaneda or Macchiaevello methods.
2. Glycogen matrix in C.trachomatis – Iodine staining – but
weak or low sensitivity.
b. Immunofluorescence: FITC labeled mAb against
species specific or genus specific Ag of C.trachomatis is
used.
 -Genus specific, also detect C.psittaci, C.pneumoniae.
 -Sensitivity and specificity 90% and 95%.
 -It is rapid method done under 1 hour.
2. ELISA for chlamydial antigens : Detection of soluble genus
specific antigen captured by antibody attached to a solid
surface eg. Plastic bead or micortiter well and later detected
with enzyme labeled detector system and chromogenic
substrate.
 Sensitivity and specificity similar to IF.
 3. DNA probes/ NAAT:
DNA hybridization can be used for direct detection of
C.trachomatis antigen in conjunctival and cervical smears.
 PCR – Polymerase Chain Reaction – common endogenus
plasmid DNA, the ompl gene [codes for MOMP] and the 16s
rRNA gene can be amplified and detected by PCR.
- This method is more sensitive than culture.
4.Chemilumnescence assay –
- acridium – ester labeled ss DNA probe which is
complementary to RNA of C.trachomatis is used.
- The labeled DNA, RNA hybrid is detected in a
luminometer.
- Luminometer measures the light emitted by the acridium
ester label.
- Sensitivity and specificity 95%.
CULTURAL CHARACTERS: Imp. for laboratory
diagnosis.
Introduction of the specimen–
 1. Intranasal, intraperitoneal, intraurethral inoculation
into mice.
 2. Into yolk sac of 6-8 days old chick embryo
 3. Cell cultures.
a. Mice will die within 10 days. Smears taken from lung,
peritoneal exudates, spleen or brain show EBS.
b. Yolk sac – organism here multiply in the endothelial
cells. Detected by making impression smears – Stained
by Giemsa, Machivello, Gimenez, C. trachomatis grow
at 35oC, C.psittaci at 39oC
c. Cell culture – Cells have to be irradiated or treated
with metabolic inhibitor for isolation. Cycloheximide
treated McCoy cells most commonly used.
C.pneumoniae grow better in HeLa cells or Monkey
Kidney cell lines.
 Mouse fibroblast cells can also be used.
 C. psittaci can be propagated in fish or lizard cells.
 Presence of organism – detected by staining for
inclusions or EBs by IF or Giemsa.
6. Isolation of chlamydia – specimen inoculated in mice,
yolk sac of 6-8 day chick embryo, cell culture.
7. Detection of chlamydial antibody: Antibody against
C.psittaci and serovars L1 – L3 of C.trachomatis can be
detected by CF test.
 Ab against C.trachomatis and C.pneumoniae can be
detected by micro immunofluorescence using EB’s of
standard serovars. Also can use immunoperoxidase and
ELISA test. High level of IgM [>1:64] and rising titer of
IgG diagnostic.
Treatment
 Trachoma - Azithromycin id DOC, Erythromycin &
Doxycycline
 Genital infections & Inclusion conjunctivitis:
Doxycycline & Azithromycin
 LGV : Doxycycline & Sulfonamides
 Respiratory tract infections: tetracycline or
erythromycin
Prophylaxis
 Trachoma -> Personal hygiene
 Genital infections ->
- Detection of cases, contacts & their treatment
- Psittacosis ->
- Avoiding contact with infected birds
- Elimination of infected birds