The History of Discovery
Primary discovery of chlamydial inclusion bodies in conjunctival exudate of patient with trachoma was made in 1907 by S. Prowazek and L. Halberstädter. They found microbial microcolonies later termed as Halberstädter-Prowazek bodies enwrapped within common coat in the cytoplasm of infected cells. Hence, these and other similar bacteria were termed “Chlamydia” (from Gr. chlamyda that means “cloak”).
Classification of Chlamydiae
- The order Chlamydiales includes the family Chlamydiaceae; pathogenic represenatives pertain to genera Chlamydia and Chlamydophila.
- Human pathogen Chlamydia trachomatis causes trachoma, lymphogranuloma venereum or Nicolas-Favre disease, inclusionconjunctivitis, and numerous nongonococcal urogenital infections like urethritis and salpingitis.
- The genus Chlamydophila comprises two species pathogenic for humans – C. pneumoniae and C. psittaci.
- C. pneumoniae causes human pneumonia, bronchitis and sinusitis, whereas C. psittaci is the causative agent of avian disease ornithosis (or psittacosis) that in some cases may occur as a severe respiratory infection in humans.
Structure and Properties of Chlamydiae
Morphology and life cycle
- Chlamidiae are obligate intracellular parasites. They are non-motile and non-sporeforming.
- Bacteria are of very small sizes and have two stages in life cycle – elementary bodies and reticulate bodies.
- Elementary bodies measuring 0.2-0.3 μm possess infectious properties being capable of invading the host cells.
- In the infected cells elementary bodies transform into vegetative reticulate inclusions 0.8-1.5 μm in size. They might be covered with capsule. After several reproductions reticulate bodies convert again into elementary invasive forms.
- The whole developmental cycle takes about 48-72 hours.
- According to their structure, chlamydiae are gram-negative bacteria with atypical peptidoglycan without acetylmuramic acid but with multiple cystein-containing peptide cross-bridges.
- Chlamydiae are primarily visualized by Romanowsky-Giemsa stain (reticulate bodies produce blue inclusions attached to cell nuclear membrane, while elementary bodies stain purple). Intracellular detection of bacteria is also performed by immunofluorescence technique.
- As the obligate intracellular parasites, chlamydiae grow in cultures of a variety of eukaryotic cell lines.
- McCoy cells are commonly used to isolate these pathogens. All types of chlamydiae proliferate in embryonated eggs, particularly in the yolk sac. Various animal models are used also for cultivation, e.g. mice.
- In general, chlamidiae render weak biochemical activity. Bacteria are unable to synthesize ATP and need the host cell for energy and nutrient donations.
- Some chlamydiae demonstrate endogenous metabolism like other bacterial representatives. They can liberate CO2 from glucose, pyruvate, and glutamate; they also contain dehydrogenases.
- Chlamydiae possess group-specific antigens. These are heat-stable lipopolysaccharides.
- Serovar-specific antigens are mainly outer membrane proteins (OMP). Major outer membrane protein (MOMP) covers about 60% of total amount of proteins in chlamydial cells. Other protein antigens of microbial outer membrane are variable (Pmp, OmcA, OmcB and others).
- Antigenic proteins are also found in the coat encasing bacterial intracellular inclusions (Inc proteins).
- Specific antigens are shared by only a limited number of chlamydiae. Fifteen serovars of С. trachomatis have been identified (e.g., A, B, Ва, C; D-K; L1-L3).
- Virulence factors of chlamydiae are not completely elucidated.
- Microbial LPS displays proinflammatory properties as endotoxin.
- The proteins of outer membrane such as MOMP are the bacterial adhesins. Together with cystein-containing chlamydial proteins they suppress phagocytosis inhibiting phagosome-lysosome fusion.
- Heat shock proteins hsp60 and others stimulate cellular inflammation.
- Chlamydiae possess type III secretion system (T3SS) with activity of injectisome. The structures of T3SS are responsible for microbial invasiveness and intracellular persistence.
- For instance, effector protein TARP after injection into the cell stimulates cytoskeleton remodelling and next membrane folding. It leads to engulfment of attached bacteria and their entry into the epithelial cells.
- Another effector protein CPAF with proteolytic activity destroys intracellular regulatory proteins, thus preventing the apoptosis of infected cells and presentation of chlamydial antigens.
- In general, chlamydiae demonstrate a low environmental resistance. More stable are elementary bodies, which stay viable for 5-10 min within droplet aerosol phase.
- The temperatures above 40°C and pH fluctuations rapidly inactivate bacteria. Nevertheless, their survival might be longer at low temperatures and in clinical samples with high protein contents.
- Chlamydiae are sensitive to all conventional antiseptics and disinfectants.
Pathogenesis and Clinical Findings in Chlamydial Urogenital infections
- Humans are natural hosts for С. trachomatis. The bacterium causes various human infections depending on the microbial serovar.
- Serovars А, Ва, В and С are the agents of trachoma; serovars from D to K are responsible for urogenital infections, and L-1, L-2, L-3 serovars cause lymphogranuloma venereum.
- Trachoma is an ancient eye disease. It is a chronic keratoconjunctivitis that begins with acute inflammatory changes in the conjunctiva and cornea and progresses to scarring and blindness.
- Also С. trachomatis cause numerous urogenital infections. Bacteria of serovars D-K cause sexually transmitted diseases and may also produce the specific infection of the eyes (inclusion conjunctivitis).
- The bacteria bind to epithelial cells by multiple adhesins, enter the infected cell by the action of type III secretion system and impair normal cellullar metabolism.
- Chlamydia persistence stimulates chronic inflammatory reactions within urogenital tract that may lead to sclerosis.
- Propagation of chlamydiae followed by the egress of elementary bodies by lysis or membrane body extrusion results in degradation of urogenital epithelium.
- In males С. trachomatis provokes nongonococcal urethritis and epididymitis. In females С. trachomatis causes urethritis, cervicitis, and pelvic inflammatory disease, which can lead to sterility and predisposes to ectopic pregnancy.
- Up to 50% of nongonococcal urethritis in men or the urethral dysuria in women is associated with chlamydiae. Overall, С. trachomatis annually causes more than 140 mln cases of sexually transmitted infections worldwide.
- The infection may stay long asymptomatic but transmissible to other persons.
- The newborns acquire the chlamydial infection, when passing through the infected maternal birth canal. From 20 to 50% of newborns may acquire the infection, 15-20% of them display eye symptoms and 10-20% demonstrate the involvement of respiratory tract.
- Inclusion conjunctivitis of the newborns commences as suppurative conjunctivitis arisen in 1-2 weeks after the delivery. It is manifested like chronic chlamydial infection similar to childhood trachoma.
Laboratory Diagnosis of Chlamydial Urogenital Infections
- A cytology brush or swab is used to detach epithelial cells 1-2 cm deep from the endocervix. A similar method is applied to collect specimens from the vagina, urethra, or conjunctiva. Biopsy specimens of the uterine tube or epididymis can also be examined.
- The presence of chlamydia inclusions in smears is determined by microscopy with Romanowsky-Giemsa stain and immunofluorescence microscopy.
- The swab specimens should be placed into chlamydia transportation medium and kept at refrigerator temperature before transportation to the laboratory.
- McCoy cells grown in monolayers are inoculated for culture. The inoculum from the swab specimen is incubated at 37°C for 48-72 hours.
- The monolayers are examined by direct immunofluorescence to visualize the cytoplasmic inclusions. This method of chlamydial cultures demonstrates about 80% sensitivity and near 100% specificity.
- Nevertheless, cultural tests remain laborious and cumbersome. Therefore, the laboratory diagnosis of chlamydial infections in clinical practice is mainly based on PCR as the highly sensitive, specific and reproducible molecular genetic test.
- It is more sensitive than culture and other nonamplification tests. The specificity of PCR appears to be close to 100%.
- Direct fluorescent antibody assay and enzyme-linked immunoassay (ELISA) are used to detect C. trachomatis by their antigens.
- Serological diagnosis of chlamydial infections (e.g., by ELISA) indicates the growth of serum antibodies against the pathogen.
- The rise of levels of specific antibodies occurs during and after the acute chlamydial infection.
Treatment of Urogenital Chlamydioses
- Macrolides and azalides (e.g., azithromycin) are commonly used for treatment of urogenital chlamydial infections.
- Erythromycin is given to pregnant women. Tetracyclines (e.g., doxycycline) can be administered as well.
- Aminoglycosides and β-lactams are clinically inefficient due to the poor availability of chlamydiae inside the cells.
- Topical tetracyclines or macrolides are administered in case for inclusion conjunctivitis, sometimes in combination with a systemic drug.
- Efficient vaccines for prevention of chlamydial infections are not yet elaborated.