The Chlamydia

Morphology and Identification

General
Infectious species:
Differentiated by: antigenic
composition, intracellular
inclusions, sulfadiazine
susceptibility, and disease
productions
§ systems → entry via receptor-mediated endocytosis in clathrin-coated pits or pinocytosis
Typical Organisms
§ Similar in morphologic
features, share a common
group antigen, multiply in the
cytoplasm of their host cells
via a distinct cycle
§ Obligate Intracellular
Pathogens – lack the
mechanism for production of
metabolic energy
Developmental Cycle Structure and Chemical Composition
Environmental Body (EB) § Outer cell wall: similar to gram-neg
§ Environmentally stable infectious particle aka transmissible form § Relatively high lipid content – including lipopolysaccharide of
§ Diameter: 0.3 μm; electron-dense nucleoid low endotoxic activity
§ Membrane proteins – highly cross-linked § Rigid but does not contain a typical bacterial peptidoglycan
§ High affinity for host cells § Important structural component, MOMP – encoded by ompA
§ Entry: interaction between other membrane proteins and heparin sulfate proteoglycan of w MOMP antigenic variants of C trachomatis – associated
the host cells → additional and irreversible binding to a variety of other host cell receptors with different clinical syndromes
(via multiple adhesisn: OmcB, Majr outer membrane protein, glycosylated MOMP, and § Penicillin-binding proteins are present; thus cell wall
other) → Entry is done via cytoskeletal rearrangement, activation of type III secretion formation is inhibited by penicillins and other drugs that
inhibit transpeptidation of bacterial peptidoglycan
via noncoated pits → lysosomal dusion is inhibited → protected membrane bound § Lysozyme – no effect on chlamydial cell walls
environment is made around chlamydiae → disulphide bond of EB membrane proteins § N-acetylmuramic acid – absent from cell walls
are reduced → reorganized into RB § DNA and RNA are present in both EB and RB
§ Attached near the base of microvilli where they are engulfed by host cells w EB: DNA = RNA; RB: 4DNA = RNA
Reticular Body (RB) w EB – DNA @ nucleoid; RNA @ ribosomes
§ Replicative form w Circular genome: 1.04 Mbases; encodes 900 genes; one
§ Diameter: 0.5 1 μm; devoid of electron-dense nucleoid of the smallest bacterial genomes
§ Within membrane-bound vacuole: RB grows in size and divides repeatedly via binary § asdas
fission → vacuole is filled with EBs → cytoplasmic inclusion → liberated from host cell to
infect new cells

Cycle time: 48 – 72 hours

Staining Properties
Giemsa stain
§ EM bodies – Purple
§ Host cell cytoplasm – blue
§ RB – Blue
§ Fully formed, mature intracellular
inclusions of C trachomatis –
compact masses near nucleus –
dark purple
Gram stain
§ Negative or variable; not useful
Immunofluorescence
§ Particles and inclusions stain
brightly
Dilute Lugol’s iodine solution
§ Some inclusions of C
trachomatis appear brown
because of the glycogen matrix
that surround the particles
§ C psittaci inclusions – diffuse
intracytoplasmic aggregates
Antigens Growth and Metabolism
§ Shared group (genus) – specific antigens § Require an intracellular habitat because of the
§ Heat stable lipopolysaccharides with 2-keto-3- small genome size
deoxyoctanoic acid as immunodominant component § Dependent upon host for development and
§ Antibodies to these antigens – detected by Complement energy requirements
Fixation and Immunofluorescence § Grow in cultures of a variety of eukaryotic cell
§ Species- or serovar-specific antigens – mainly outer lines
membrane proteins § McCoy cells with cycloheximide – isolate
§ Specific antigens – via immunofluorescence using chlamydia
monoclonal antibodies § C pneumoniae – HL or Hep-2 cells
§ C trachomatis – 15 serovards; separated into 2 biovariants § All types – embryonated eggs, particularly in
→ different clinical syndromes the yolk sac
w Trachoma biovar – serovars A, B, Ba, C + gental tract § Some – endogenous metabolism
serovars D – K w Liberate CO2 from glucose, pyruvate, and
w Lymphogranuloma venereum biovar – serovars L1, L2, glutamate
and L3 w Contain dehydrogenases
§ C pssitaci – serovars can be demonstrated by CF and § Replication – inhibited by many antibacterial
microimmunofluorescence (MIF) tests drugs.
§ C pneumoniae – only 1 serovar has been described w Cell wall inhibitors → morphologically
defective forms; not effective as treatment
w Inhibitors of protein synthesis (tetracyclines,
erythromycins) → effective in most clinical
infections
w Inhibited by Sulfonamides → C trachomatis
strains that synthesize folates
w Aminoglycosides – non-inhibitory
Characteristics of Host-Parasite
Relationship
§ Subclinical infection is the rule,
overt disease the exception
§ Spread from one species to another
→ frequently leads to disease
§ Antibodies to several antigens are
regularly produced but have little
protective effect against reinfection
§ Infectious agant – can persist
despite high antibody titers
Treatment
§ With effective antimicrobial drugs,
prolonged periods → eliminate
chlamydiae from infected host
§ Very early, intensive treatment →
suppress antibody formation
§ Late treatment in moderate doses
→ suppress disease but infective
agent persists in tissue
Immunization
§ Unsuccessful in protection against
reinfection
§ Prior immunization → milder
disease upon reinfection but at
times, accompanying
hypersensitivity aggravates
inflammation and scarring

Specific Organisms
 Chlamydia trachomatis Chlamydia pneumoniae Chlamydia pssitaci

Inclusion Morphology Round, vacuolar Round, dense Large, variable shape, dense

Glycogen in Inclusions Yes No No

EB Morphology Round Pear shaped, round Round

Susceptible to
Yes No No
Sulfonamides
Plasmid Yes No Yes

Serovars 15 1 ≥4

Natural Host Humans Humans, Animals Birds

Mode of Transmission Person to person, mother to infant Airborne person to person Airborne bird excreta to humans
Respiratory tract infections: Pneumonia, bronchitis,
Major Disease Trachoma, STDs, Infant pneumonia, LGV Psittacosis, pneumonia, fever of unexplained origin
pharyngitis, sinusitis
Chlamydia trachomatis

Serovar A, B, Ba, C D–K LGV biovar L1, L2, and L3

Infection Ocular Respiratory Genital

Disease Trachoma Inclusion Conjunctivitis Neonatal Pneumonia STDs Lymphogranulum venereum

§ Ancient eye disease, described in § 10 – 20% of newborns may § Suppurative inguinal adenitis
Ebers Papyrus from Egypt 3800 develop § Common in tropical climates
years ago. § 2 – 12 weeks after birth → Properties of the Agent
§ Chronic keratoconjunctivitis pneumonia § Particles – CF heat-stable
Remarks
§ Begins with acute inflammatory chlamydial group antigens
changes in the conjunctiva and
cornea → scarring and blindness

§ Incubation period: 3 – 10 days
§ Endemic areas: infection occurs
in early childhood
§ Often mixed with bacterial
conjunctivitis
§ Earliest symptom: lacrimation,
mucopurulent discharge,
conjunctival hyperemia, and
follicular hypertrophy
§ Microscopic examination of
cornea: epithelial keratitis,
subepithelial infiltrates, extension
Clinical Findings
of limbal vessels into the cornea
(pannus)
§ As pannus extends downward:
scarring of the conjunctiva, eyelid
deformities, trichiasis
§ With 2˚ bacterial infection: loss of
vision progresses over a period
of years
§ No systemic symptoms or signs
of infection
Adults
§ Genital secretions of infected
adults → self-inoculated into
conjunctiva
Newborns
§ Acquired via infected birth
canal
§ Starts as a mucopurulent
conjunctivitis; 5 – 12 days old
§ Subsides with erythromycin
or tetracycline treatment OR
spontaneously after weeks
§ Nasal obstruction or discharge
§ Absence of fever
§ Eosinophilia
§ Radiographs – interstitial
infiltrates and hyperinflation
Men
§ Nongonococcal urethritis
§ Epididymitis
Women
§ Urethritis, Cervicitis
§ Pelvic inflammatory disease
w Can lead to sterility and
predisposes them to etopic
pregnancy
Men and Women
§ Proctitis and proctocolitis
§ More common in men who have sex
with men
Nongonoccocal urethritis (M) and
urethral syndrome (W)
§ Dysuria, nonpurulent discharge,
increased frequency of urination
§ Genital secretions of infected adults
can be self-inoculated into the
conjunctive → inclusion
conjunctivitis (similar to trachoma)
§ First: small, painless, and
evanescent papule or vesicle @
area of contact → may ulcerate OR
unnoticed and heals → Later (days
– weeks): regional lymph nodes
enlarge, become matted, and
painful
§ Men: inguinal lymph nodes above
and below Poupart’s ligamn → skin
on top becomes purplish →
suppurate (bubo formation) →
discharge pus via sinus tracts
§ Women and homosexual Men:
perirectal nodes with proctitis, and
a bloody mucopurulent anal
discharge
Active lymphadenitis
§ Systemic symptoms: fever,
headaches, meningismus,
conjunctivitis, skin rashes, nausea,
vomiting, and arthralgias

Untreated

Culture
§ Typical cytoplasmic inclusions in
epithelial cells of conjunctival
scrapins – stained with
fluorescent antibody or Giemsa
method; occur at early stages of
disease and on the upper tarsal
conjunctiva
§ Innoculation of scarpings into
cycloheximide-treated McCoy cell
cultures → growth
Serology
§ Infected individuals – develop
both group anitbodies and
serovar-specific antibodies in
serum and eye secretions; does
not confer significant resistance
to reinfection
§ Immunofluorescence – most
sensitive method of detection
Molecular Methods
Laboratory
§ Only research projects have used
Findings/Diagnosis
PCR
§ Mass Azithromycin treatment →
infection and clinical disease are
decreased at 6 and 12 months
Treatment therapy
§ Azithromycin ≫ erythromycin and
doxycycline
§ Topical therapy is of little value
§ Topical tetracycline or
erythromycin for neonatal N.
gonorrhoeae but not in C.
trochomatis
§ Systemic therapy for
inclusion conjunctivitis
§ Oral erythromycin for 14 days
§ Systemic erythromycin – severe
cases
Specimen Collection
§ Specimen should contain infected
human cells + extracellular material
where they might be present
§ Endocervical specimens (after
discharge and secretion removal)
§ Collect with dacron, cotton, rayon on
a plastic shaft
§ First 20 mL of voided Urine can be
used for nucleic acid detection.
§ Swab specimen – kept in a
chlamydiae transport medium – 2
sucrose phosphate supplemented
with bovine serum and antibiotics –
kept at refrigerator temp
Molecular Methods
§ Nonamplified probe assay – 104
copies of the 16S rRNA
§ Nucleic acid amplification tests –
test of choice for diagnosis; uses
cryptic plasmid or 23SrRNA
Direct Cytologic Examination and
Enzyme-linked Immunoassay
§ DFA: Monoclonal antibodies
targeting specific-specific antigen on
chlamydial MOMP
§ EIA: genus specific antigens from
EBs
§ DFA > EIA
Culture
§ Susceptible cell lines – McCoy,
HeLa 229, HEp-2 – grown
monolayers on coverslips in dram or
shell vials; treated with
cycloheximide to inhibit metab and
increase sensitivity of isolation
§ ~80% sensitive; 100% specific
Serology
§ Not generally useful
§ Tetracyclines are used
(requirement: NOT pregnant)
§ Azithromycin
§ Inflammatory region → fibrosis →
lymphatic obstruction and rectal
strictures
§ Lymphatic obstruction →
elephantiasis of penis, scrotum, or
vulva
§ Chronic proctitis → progressive
rectal strictures, rectosigmoid
obstruction, and fistula formation
Smears
§ Pus, buboes, biopsy material – may
be stained but particles are rarely
recognized
Nucleic Acid Amplification Tests
§ Can detect LGV serovars but
cannot differentiated from other C
trachomatis serovars
Culture
§ McCoy cell cultures
§ Inoculum – treated with an
aminoglycoside to lessen bacterial
contamination
§ Agent identification – morphology
or serologic tests
Serology
§ Antibodies via complement
formation reaction
§ Positive: 2 – 4 weeks after onset of
infection
§ Early: Sulphonamides and
tetracyclines
§ Late stages: surgery
 Epidemiology Prevention Epidemiology Epidemiology
§ 400 million have trachoma § Neonatal IC – diagnosis and § Sexual contant with infected sex § Highest incidence: subtropical and
§ 20 million are blinded by it treatment of the pregnant parteners tropical
§ Most prevalent: sub-Saharan woman and her sexual § Neonatal IC – mother’s genital tract § Occurs all over the world
Africa, Asia, Mediterranean partner § Spread: sexual contact
basin, where hygienic conditions Prevention § Portal of entry: eye, genitals
are poor and water is scarce § Annual screening 25 years and § Reservoirs of infection: genital
§ Hyperendemic areas: childhoos younger tracts and rectums of chronically
infection may be universal and infected individuals
severe blinding disease is § Lab personnel exposed to aerosols
common → chlamydial pneumonitis with
§ US, trachoma occurs sporadically mediastinal and hilar adenopathy
in some areas and endemic foci
Epidemiology and Control persist
Control
§ SAFE program
§ Surgery for deformed eyelids
§ Periodic Azithromycin therapy
§ Face washing and hygiene
§ Environmental improvement
(building latrines, decreasing the
number of flies that feed on
conjunctival exudates
§ Improved socioeconomic
conditions enhance the
disappearance of endemic
trachoma
Chlamydia pneumoniae Chlamydia psittaci
§ First strain – 1960s from chick embryo yolk sac culture § Psittacosis – applied ot the human C psittaci disease acquired from contact with birds and
Remarks also infection of psittacine birds
§ Ornithosis – infection with similar agents in all types of domestic birds and free-living birds
§ Round, dense, glycogen-negative inclusions § Propagaed in embryonated eggs, in mice and other animals, and some cell cultures.
§ Sulfonamide resistant
Properties of the Agent § EBs – pear-shaped appearance
§ Only 1 serovar

§ Mostly asymptomatic or with mild illness (severe cases have been reported tho) Pathogenesis and Pathology
§ No SSx that differentiate it § Entry: Respiratory tract
§ Upper and lower airway diseases occur § Found in the blood during first 2 weeks
§ Pharygitis is common § Found in sputum by the time lungs are involved
§ Sinusitis and otitis media may occur with a lower airway disease § Psittacosis – patchy inflammation of the lungs in which consolidated areas are sharpy
§ An atypical pneumonia similar to that caused by Mycoplasma pneumoniae is the primary demarcated
Pathogenesis and
recognized illness. § Exudates – mononuclear ≫
Pathology
§ Lesions are similar to those in pneumonitis
OR
§ Liver, spleen, heart, and kidney – enlarged and congested
Clinical Findings
Clinical Findings
§ Sudden onset of illness in the form of influenza or non-bacterial pneumonia in a person
exposed to birds
§ Incubation period – 10 days
§ Onset: sudden, can be insidious, with malaise, fever, anorexia, sore throat, photophobia, and
severe headache
Smears Culture
§ Fluorescent antibody techniques – insensitive § Detection via immunoassays or PCR is preferred
Culture § Can be cultured from blood, sputum, or lung tissue
§ Swab specimens of pharynx → chlamydiae transport medium @ 4˚C § Cultured on tissue culture cells, embryonated eggs, or mice
Laboratory Diagnosis
§ HL and HEp-2 cells > HeLa 229 or McCoy § BSL 3
§ Growth is better at 35˚C than 37˚C § Isolation is confirmed: serial transmission, microscopic demonstration, serologic identification
§ Addition of cycloheximide – increases sensitivity inhibits eukaryotic cell metab Antigen Detection of Chlamydia psittaci (in reference or research labs)
§ DFA staining
 § After 3 days: cells are fixed and Cpneuminae-specific monoclonal antibody conjugated with § Immunoassay
fluorescein § Molecular diagnosis using PCR
Serology Serology
§ Serology using MIF test → most sensitive; species specific § Complement fixing
w Primary Infection: IgM – 3 weeks → IgG – 6 – 8 weeks § Microimmunofluorescent antibodies
w Reinfection: IgM – minimal or absent → IgG – 1 – 2 weeks
Nucleic Acid Amplification Methods
§ BioFire Diagnostics, Inc. received FDA approval for addition of C. pneumoniae FilmArray
Respiratory panel
§ Prolonged infections can occur § Incomplete
Immunity § Asymptomatic carriage may be common § Carrier state – persists for 10 years after recovery

§ Susceptible to macrolides and tetracyclines and to some fluoroquinolones § Doxycycline and tetracyline is preferred
§ Treatment with doxycycline, azithromycin or clarithromycin, levoflaxin or moxifloxacin → § Macrolides and fluoroquinolones – alternatives
Treatment
beneficial
§ Course: 10 – 14 days
§ Worldwide § Birds kept as pets – important source of human infection
§ Infection: epidemic and endemic § Pigeons
Epidemiology § No known animal reservoir, transmission is presumed to be from person to person
§ Predominantly airborne
§ Controversial link to coronary artery disease