Chlamydia : Chlamydia Mr. Gunjal Prasad Niranjan M.Sc. Medical Microbiology PG Dip. CR & RA Assistant Prof. Dept. Microbiology PDVVPF’s Medical College, Ahmednagar Introduction : Introduction Obligate intracellular parasites. Small, Non - motile, Gram negative. Have tropism for squamous epithelial cells & macrophages of respiratory and gastrointestinal tracts. Cause 1. Trachoma, 2. Lymphogranuloma Venerum (LGV), 3. Psittacosis. Can cause diverse diseases in birds and mammals. The name Chlamydia – Derived from characteristic appearance of inclusion body by these agents. The inclusion bodies enclose the nuclei of infected cells as a cloak or mantle ( Chlamys meaning =mantle = symbol of authority). Difference between Chlamydia and Viruses : Difference between Chlamydia and Viruses Are intercellular parasites, so previously thought to be viruses. Also they are filterable through filters retaining bacteria. Also due to failure to grow on cell free media. Differ from viruses on following properties - Lack the ability to produce own ATP, use host cell ATP. Hence called Engery parasites. They have both DNA and RNA as bacteria. Rigid cell walls and ribosomes as bacteria. Multiply by binary fission. Susceptible to antibiotics and chemotherapeutic agents. They do not have an eclipse phase following cellular infection like viruses. CHLAMYDIA: CHLAMYDIA Order – Chlamydiales Family – Chlamydiaceae (only one family) 4 species in the Genus Chlamydia – C. trachomatis, C. pneumoniae, affects humans C. psittaci and C. pecorum affects ruminants All are non-motile, Gram Negative; share antigens, have both DNA and RNA. C. trachomatis : C. trachomatis 20 serotypes, classified on the basis of neutralization and Immunofluorescence tests. A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, Ja, K, L1, L2, L2a, L2b & L3. Serotypes A, B, Ba, C – causes Hyperendemic Trachoma – causes inflammation of the conjunctiva and cornea of the eye and the formation of scar tissue . Serotype D, Da, E, F, G, H, I, Ia, J, Ja, K – Cause Inclusion Conjunctivitis, Non - Gonococcal Urethritis (NGU), Salpingitis – inflammation of fallopian tube, cervicitis, Pneumonia of new born. C. trachomatis : Serotypes L1, L2, L2a, L2b, & L3 – cause Lymphogranuloma Venerum. (LGV). - Disease caused by a species of Chlamydia bacterium; transmitted by sexual contact; Causes inflammation of and prevents drainage of the lymph nodes in the genital area. LGV can cause destruction and scarring of surrounding tissue. C. trachomatis C. pneumoniae : C. pneumoniae Its an exclusive human pathogen with no animal host. Causes – Acute Respiratory Disease in humans. Has only 1 serotype. A. Ocular Infection – Trachoma : A. Ocular Infection – Trachoma Caused by C. trachomatis serotype A, B, Ba, & C Chronic Kerato-conjunctivities. Major cause of blindness. Characterized by follicles, papillary hyperplasia, pannus formation and in the late stage – Cicatrization. Transmission – Eye to eye through finger or contaminated towel, clothing. Incubation period – 3-10 days. In endemic area children below 9 yrs age are mostly affected. Trachoma has been characterised into I – IV stages, early stage is most infective one. 2.Inclusion Conjunctivitis : 2.Inclusion Conjunctivitis Caused by C. trachomatis serotype D to K. Prevalent in sexually active young people & spread from genital secretions by hand to eye. This is formerly known as “ Swimming pool conjunctivitis” as it was associated with swimming in contaminated water. Also know as Paratrachoma. 3. Opthalmia neonatorum : 3. Opthalmia neonatorum Neonatal form of Inclusion conjunctivitis. Infants acquires infection during passage through the infected birth canal. It usually becomes apparent between 5-12 days after birth. About 20-50% infants of infected mothers develops the infection. B. Genital Infections Genital Chlamydiasis : B. Genital Infections Genital Chlamydiasis Chlamydia trachomatis causes “Genital Chlamydiasis” and LGV. Both are sexually transmitted diseases. 1. Genital Chlamydiasis - Caused by D to K serotype. Responsible for 40% of cases of NGU, a STD. In males in addition to urethritis also causes – Epididymitis, Proctitis. In females urethritis, cervicitis, salpingitis and Pelvic Inflammatory Disease. Infection may symptomatic or asymptomatic. Symptoms – Dysuria, non-purulent discharge and frequency in urination. 2. Lymphogranuloma Venereum : 2. Lymphogranuloma Venereum LGV is a sexually transmitted disease caused by C. trachomatis serotypes L1 to L3. The primary lesion consist of small painless papule or vesicle on external genitalia. It may ulcerate or heal spontaneously in a few days. The regional lymph nodes (inguinal in males and intrpelvic & para - rectal in female) are enlarged, tender, and may break open with the formation of sinuses. The enlarged inguinal lymph nodes are named bubos. C. Respiratory Infections – Pneumonia : C. Respiratory Infections – Pneumonia Commonest cause of pneumonia following S. pneumoniae & H. influenzae. It is an important risk factor in cardiovascular disease, where organism is isolated from coronary artery. However more studies are required to ascertain its role. 2. Psittacosis : 2. Psittacosis C. psittaci causes “Psittacosis and Ornithosis” in birds and man. Human infection occurs by inhalation of infected dried faeces. Psittacosis (Psittacos – parrot) is a disease of parrots. Disease acquired from non- psittacine birds known as “Ornithosis” (Ornithos – birds). Incubation period – 1-2 wks. Disease may vary from mild influenza to severe illness with pneumonia, septicaemia and meningoencephalitis. Laboratory Diagnosis: Laboratory Diagnosis 4 approaches available: Microscopic demonstration of inclusion or elementary bodies. Isolation of Chlamydia. Demonstration of Chlamydial Ag. Demonstration of Abs or hypersensitivity. 1. Microscopy : 1. Microscopy Gram stain – Gram Negative,. Stain better with Castaneda, Mchiavello, or Giemsa Stain. Inclusion bodies are basophilic & present in cytoplasm. Inclusion bodies can be stained with - Lugol’s iodine – because of presence of Glycogen matrix. Immunofluorescence staining. IF staining 2. Culture : 2. Culture Animal inoculation Yolk sac of 6 - 8 days old chick embryo . Tissue culture – McCoy, HeLa cell lines Infected cell cytoplasm has a granular appearance 2. Culture : 1. Animal inoculation – Mice are inoculated by intranasal or intraperitoneal or by intracerebral inoculations. Mice die within 10 days and smears from various tissue (lung, peritoneal exudates, spleen or brain) shows elementary bodies. 2. Yolk Sac Inoculation – Yolk sac of chick embryo is inoculated & the organism can be detected in impression smears stained by Giemsa stain. 2. Culture 2. Culture : 3. Tissue Culture – McCoy cell lines treated with cycloheximide are mostly used. Mouse fibroblast, HeLa 229 or monkey kidney cells can also be used. Organism growth is detected in tissue culture by staining for elementary bodies or inclusion bodies. 2. Culture Infected cell cytoplasm has a granular appearance Inclusion bodies: Inclusion bodies Demonstration of characteristic inclusion bodies. Trachoma: Halber staedter Prowazekii or HP bodies in conjunctival scrapings. Psittacosis: LCL (Levinthal – Cole - Lillie) bodies in alveolar macrophages, cell lines. Treatment and Prevention: Treatment and Prevention Tetracycline, erythromycin and sulfonamides. Vaccines are of little value. Treatment coupled with improved sanitation. Safe sexual practices. Treatment of patients and their sexual partners. MYCOPLASMA: MYCOPLASMA Morphology : Morphology Smallest Free living microorganisms. Can pass through bacterial filters. Pleomorphic. May present as small spherical or branching filaments. Lack cell wall but have triple layered cell membrane rich in cholesterol and other lipids. Gram negative. Better stained by Giemsa stain. Non sporing, Non-flagellated . Mycoplasma of Humans: Mycoplasma of Humans Parasitic Established pathogens : M. pneumoniae. Presumed pathogens: M. hominis , U. urealyticum. 3. Non pathogenic: M. orale, M. buccale, M. genitalium, M. fermentans. 4. Saprophytic – Present mainly on skin & in mouth. Pathogenicity : Pathogenicity Produce surface infections – Adhere to the mucosa of respiratory , gastrointestinal & genitourinary tracts with the help of adhesin. Two types of diseases: Atypical Pneumonia Genital infections Mycoplasma pneumonia: Mycoplasma pneumonia Causes Primary Atypical Pneumonia/ Walking pneumonia. Seen in all ages. Incubation period: 1-3 wks. Transmission: Airborne droplets of nasopharyngeal secretions, close contacts (families, military recruits). Mycoplasma pneumonia: Mycoplasma pneumonia Gradual onset with fever, malaise - mild sickness or depression , chills, headache & sore throat. Severe cough with blood tinged sputum (worsens at night). Complications: Bullous myringitis – Inflammation of eardrum. Otitis – Inflammation of ear, Meningitis – Inflammation of meningis, Encephalitis – Inflammation of brain, Hemolytic anemia – Abnormal breakdown of RBCs. Genital Infections: Genital Infections Caused by M. hominis & U. urealyticum. Transmitted by sexual contact. Men - Nonspecific urethritis - Inflammation of Urethra, Proctitis - Inflammation of the rectum; marked by bloody stools and a frequent urge to defecate, Balanoposthitis - Inflammation of both the head of the penis and the foreskin & Reiter’s syndrome. PowerPoint Presentation: Women – Acute salpingitis – Inflammation of fallopian tube, Pelvic Inflammatory Disease , Cervicitis – Inflammation of cervix, Vaginitis. Also associated with infertility, abortion, postpartum fever – fever immediately after birth, Chorioamnionitis – Inflammation of fetal membrane & low birth weight infants. Laboratory Diagnosis: Laboratory Diagnosis Specimens – Throat swabs, Respiratory secretions. Genital secretions. Microscopy – Highly pleomorphic , varying from small spherical shapes to longer branching filaments. Gram negative, but better stained with Giemsa. Laboratory Diagnosis: Laboratory Diagnosis Isolation of Mycoplasma ( Culture ) – Semi solid enriched medium containing 20% horse or human serum, yeast extract & DNA. Penicillin & Thallium acetate are selective agents. (serum – source of cholesterol & other lipids) Incubate aerobically for 7 -12 days with 5–10% CO 2 at 35-37 ° C. (temp range 22- 41 ° C, parasites 35- 37 ° C, saprophytes – lower temp). Laboratory Diagnosis: Laboratory Diagnosis Typical “fried egg” appearance of colonies – Central opaque granular area of growth extending into the depth of the medium, surrounded by a flat, translucent peripheral zone. Colonies best seen with a hand lens after staining with Diene’s method. 4. Produce beta hemolytic colonies , can agglutinate guinea pig erythrocytes. PowerPoint Presentation: Dr Ekta,Microbiology, GMCA Fried egg colonies Mycoplasma & HIV infection: Mycoplasma & HIV infection Severe & prolonged infections in HIV infected & other immunodeficient individuals were reported by many parts of world. Treatment: Treatment Tetracycline, Erythromycin & Clarithromycin – drug of choice. Resistant to antibiotics which interfere with bacterial cell wall synthesis. Newer Macrolides - They inhibit protein synthesis by blocking the 50S ribosomal subunit & Quinolones – Targets the bacterial enzyme DNA gyrase in order to inhibit the coiling of bacterial DNA, thus interfering with bacterial replication being used now.