Chlamydiae

Chlamydia are obligate intracellular parasite of humans, animals and birds. Based on human diseases, they were called psittacosis – lymphogranuloma - trachoma, PLT viruses agents. They lack enzymes of the electron transport chain and so require ATP and nutrient sources from the host cells. Therefore,they are called energy parasites.

General characteristics –

·    Are small, obligate, intracellular, gram negative bacteria.

·    They possess both RNA and DNA, ribosomes and cell wall similar to that of gram negative bacteria, but they lack peptidoglycan.

·    They lack the ability to produce their own ATP, hence they use host’s ATP.

·    They multiply by binary fission.

·    They are nonmotile, stain readily with Giemsa stain. Inclusion bodies are basophilic in nature. Mature inclusions of C. trachomatis possess glycogen matrix, therefore iodine stains them coppery brown. Inclusions of C. psittaci do not possess glycogen matrix, therefore they do not stain with iodine. They can be also be demonstrated by direct immunofluorescent staining.

·    They multiply in the cytoplasm of the host cell forming microcolonies or inclusion bodies which drape around the nucleus like a cloak.

4 species :

C. trachomatis - compact inclusions with glycogen matrix, sensitive to sulfonamides, natural human parasites, cause localized infections of eyes, genitals.

C. psittaci - form diffused vacuolated inclusions without glycogen matrix, resistant to sulfonamides, natural parasite of birds, animals, cause pneumonia and generalized infections in humans.

C. pneumoniae - human pathogen, cause acute respiratory disease.

Developmental cycle - Chlamydiae occur in 2 forms - the elementary body and the reticulate body.

The elementary body is the extracellular, infective form. It is a spherical particle, 200-300nm diameter, a rigid trilaminar cell wall, an electron dense nucleoid. In the absence of peptidoglycan, the rigidity of EB cell wall is due to disulpide cross-linking among MOMP (major outer membrane protein) and several cysteine- rich proteins.

The reticulate body is the intracellular, replicative form, 500-1000nm in size. Its cell wall is fragile, leads to pleomorphism.

                        Reproductive cycle diagram : refer Ananthanarayan page 390

1. Infection is initiated by the attachment of the infectious elementary body to the surface of a susceptible epithelial cell, followed by endocytosis. Inside the host cell, the elementary body lies within the endosome, being separated by the endosomal membrane, from the host cell cytoplasm. Chlamydiae – dependent modification of the endocytic membrane prevents lysosomal fusion and thus escapes degradation.
2. By 8 hours after infection, the elementary body in the endosome loses its dense DNA core, its cell wall becomes less rigid due to breaking of the disulphide bonds, increases in size, undergoes reticulation (reticulate bodies) and begins to divide by binary fission by 12 hours. It does not possess cytochrome and lacks the ability to produce ATP, but they use host’s ATP.
3. By 20-24 hrs, the reticulate bodies show central condensation, and are converted to elementary bodies. Binary fission occurs till 40 hrs. The developing chlamydial microcolony within the host cell is called the inclusion body. The mature inclusion body contains 100-500 elementary bodies, which are ultimately released. This inclusion body pushes nucleus to periphery.
4. C. psittaci damages host cell and releases the elementary bodies within 48 hours.

            C. trachomatis - inclusion bodies are exocytosed in 72-96 hours.

During the active intracellular growth of chlamydia, the organism specific lipopolysaccharides accumulate on the host surface. This highly antigenic material induces inflammatory and immunologic responses which contribute to the pathogenesis of chlamydial diseases. They are grown in the mouse, chick embryo or in cell culture. They are heat labile, inactivated at 56°C. They are susceptible to ethanol, ether, low concentrations of phenol, formalin. Infectivity maintained at 4°C.

Antigenic Properties - Chlamydiae possess 3 major kinds of antigens :

1. Heat stable, genus-specific, complement fixing, common to all chlamydiae. It is a lipopolysaccharide.
2. Species-specific protein antigens, major outer membrane protein (MOMP), present at the envelope surface.
3. Intra-species specific, located on the outer membrane proteins.

Pathogenicity –

1. Chlamydia trachomatis - causes ocular and genital infections worldwide.
• Trachoma : The disease is marked by severely swollen conjunctiva with a pebbled appearance. Scarring of the eyelid causes eyelashes to point inward, leads to the destruction of cornea and eventually blindness. Infection is transmitted from eye-to-eye by fingers or fomites. Flies may transmit the infection mechanically. Also carried by dust. The incubation period is variable and influenced by the dose of infection.

                        It is demonstrated microscopically by conjunctival scrappings, Giemsa staining or with         Lugol's iodine.They can be grown in the yolk-sac of 6-8 old eggs.

Local application and oral administration of erythromycin and tetracycline, continued for several weeks.A single dose azitromycin is effective.It is prevalent in the developing nations because of overcrowding and unhygienic conditions. Control of disease involves mass education and chemotherapy.

·         Genital chlamydiasis : Chlamydial infections has become the most common sexually   transmitted disease worldwide. In men, they cause nongonococcal urethritis, epididymitis and in women pelvic inflammatory disease. Following an incubation period of 1-3 weeks, symptoms of non gonococcal urethritis appear, ie., a scanty, watery urethral discharge. Inflammation of epididymis lead to sterility. Pelvic inflammatory disease causes infertility and ectopic pregnancy (a pregnancy in which the embryo begins to develop outside the uterus).

             Chlamydial genital infections are difficult to control.Infants become infected  while passing through the birth canal of an infected mother.Tetracycline, sulfa drugs are effective if used in both partners.

2. Chlamydia psittaci

·         Psittacosis is a disease of parrots, transmissible to human beings. A similar disease acquired by non-psittacine/other birds is ornithosis.

                        Stresses such as overcrowding, shipping to pet shops can activate the disease. Both forms        of the disease are spread by direct contact, infectious nasal droplets and faeces. Organisms found in every organ of the infected bird. The birds have diarrhoea and mucopurulent discharge from the nose and mouth. Humans usually acquire the disease from birds. Poultry workers, pet-shop owners, bird fanciers, veterinarians are susceptible. Organisms are inhaled and spread systemically to the lungs and reticuloendothelial system. After an incubation time of 1-2 weeks, onset of symptoms with sore throat, coughing, difficulty in breathing, head  ache, fever and chills.

            Diagnosis by inoculation into tissue culture. The organism can be isolated from blood, sputum. Tetracycline is the effective treatment.

3. Chlamydia pnuemoniae-   It is a common cause of respiratory disease in older children and adults.It cause pharyngitis, sinusitis, bronchitis, also associated with adult asthma. Incubation period is 1-3 weeks. Diagnosis by antigen detection.

Laboratory Diagnosis – For diagnosis of chlamydial infections ocular, urethral, vaginal and cervical specimens are best collected by scraping the mucosa. In addition, depending upon the site of involvement, blood, respiratory secretions, sputum, lung and other tissues can be collected. These specimens are processed,

1. Microscopic demonstration of  inclusion or elementary bodies - they are large enough to see under light microscope.They can be stained with Gram stain or Giemsa. Since C. trachomatis has glycogen matrix, they can be stained with Lugol's iodine. Conjunctival scrappings has been collected and stained. Other than staining, immunofluorescence can be done using monoclonal antibody. Samples are also collected from cervix, urethra.
2. ELISA for detection of chlamydial antigens – detection of genus specific antigen using immobilised antibody in a microtitre well.
3. DNA probes – DNA hybridization can be used for the direct detection of antigens.
4. PCR – sensitive method than culture.
5. Isolation of chlamydiae - By inoculation into embryonated eggs, experimental animals and tissue cultures.
6. Demonstration of chlamydial antibody-  Antibodies can be detected by complement fixation test, ELISA, immunofluorescence. A high level of IgM and a rising titre of IgG are taken as diagnostic.
7. Skin test – A skin test (Frei’s test ), a heat inactivated lymphogranuloma venerum type is grown in yolk sac of embryonated egg is injected intradermally on the forearm and a control prepared from uninfected yolk sac on the other forearm. A positive reaction is indicated by an inflammatory nodule appearing on the test arm in 2 days and reaching a maximum in 4-5 days, of 7mm diameter.

Treatment & Prophylaxis – For the treatment of trachoma, tetracycline is given topically as well as systemically for several weeks. In young children, erythromycin given. In case of ophthalmia neonatorum, erythromycin may be given orally and topically. Genital infections treated with tetracycline.

            Endemic trachoma control depends on increasing the living standards. Psittacosis has been controlled by checking the import of birds. No successful vaccine is available.