Diagnostic microbiology lecture: 16 Treponema pallidum Abed ElKader Elottol MSc. Microbiology Abed ElKader ElOttol

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General Characteristics of the Organism: 1. A thin, motile spiral organism. 2. Can not be cultivated on artificial media. 3. Can not be visualized by light microscope 4. Rabbits could be infected with this organism. Are spiral flexible organisms that move without flagella Multiply by transverse binary fission. 3Abed ElKader ElOttol

Clinical description of the disease: Syphilis is a chronic granulomatous disease that characteristically progress by stages of clinically apparent infections. Each stage has different manifestation and lesion morphology, and stages are separated by periods of latency. The only evidence of infection is a reactive serologic test during the latency periods. Except for congenital syphilis which is transmitted from infected mothers to the fetus across the placenta, syphilis is almost always transmitted by sexual exposure. 4Abed ElKader ElOttol

PRIMARY STAGE: The primary syphilitic lesion, the chancre, appears. The chancre is most frequently a painless, ulcer-like lesion with raised borders and a necrotic base. The serum (exudate) from the chancre contains T. pallidum and is highly infectious. The chancre heals spontaneously without scaring over a period of 2-3 weeks. SECONDARY STAGE: It occur 6 weeks to 6 months after the chancre heals. Symptoms of this stage includes, headache and malaise, general lymphoadenopathy and a variety of skin rashes, hepatitis, meningitis, glmerulnephritis and nephritis. Nephrosis is a result of Antigen antibody complexes deposited in the kidney. Secondary lesions heal spontenaneously over a period of several weeks and then enters in a latent stage number 2. this stage, bone and teeth deformation may occur. 5Abed ElKader ElOttol

TERTIARY STAGE: The destructive stage of the disease. It may begin 5-20 years after the initial infection. Only one third (1/3) of untreated patient develop tertiary which are of three types: 1. 80% are aneurysms () : Leading to dilatation of aortic valve) 2. 15% are CNS disease and meningitis. 3. 5% gummas (Lesions which histologically resemble a tubercle). At this stage, bone and teeth deformation may occur. CONGINITAL SYPHILIS: The fetus may be aborted, stillborn or liveborn or without clinical evidence of syphilis. The lesions of early congenital syphilis are of the secondary type and may be present at birth. Blindness secondary to optic atrophy. Deformed bones may also occur. 6Abed ElKader ElOttol

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LABORATORY IDENTIFICATION: T. pallidum can not be cultivated in the laboratory In-Vitro. Only two methods are available for the diagnosis of T. pallidum. 1. DARK-FIELD MICROSCOPY: Most specific for infectious syphilis. Clean the chancre with gauze moistened with sterile sodium chloride solution,Dry it. Scrap the edges of the chancre several times with the flat side of a sterile lancet, Do not draw blood. Press with dry sterile gauze. Remove the swab and wait for a few minutes until a pinkish serous fluid appear, Draw off the fluid with a Pasteur pipette. Place a drop of the fluid on a thin glass slide. Examine under the microscope using dark field condenser. Search for the characteristic morphology of T. pallidum. (Motile thin spirochetes, rotating around their long axes). 9Abed ElKader ElOttol

2. SEROLOGICAL TESTING: Two types of antigens are used. A. Non-Treponemal Antigens Test: It utilizes "cardiolipin" as its antigen, which reacts with IgM or IgG immunoglobulin known as "reagin" antibody 1.Flocculation test (VDRL): (Venerial Disease Research Laboratories) This test is based on the fact that particles of the lipid antigen ( cardiolipin) remains dispersed in normal sera, but combines with reagin to form visible aggregate within a few minutes. Rapid Plasma Reagin test (RPR) is a good modification for rapid surveys. 10Abed ElKader ElOttol

2. Complement fixation test "Wasserman, Kolmer" This test is based on the fact that, reagin-containing sera fix complement in the presence of cardiolipin. NB: Biologic false results may occur with many patients having: 1. Malaria 2. Leprosy 3. Measles 4. Infectious mononucleosis 5. SLE 11Abed ElKader ElOttol

B. Treponemal Antibody Test: 1. Fluorescent Treponema Antibody Absorption Test (FTA-ABS) This test employs indirect immunofluorescence (Killed T. pallidum + patient`s serum + labeled anti-human gammaglobulins). The test serum is added to the antigen (killed T. pallidum) which is fixed on a glass slide, incubated and then washed to remove excess serum. Conjugate (Labeled antihuman gammaglobulin) is added, incubated and then washed. This test is read microscopically with an ultraviolet light source. 2. T. pallidum immobilization test (TPI): Specific antibodies in the patient serum after the second week of infection could be demonstrated by their ability to immobilize actively motile T. pallidum, extracted from the chancre of infected rabbits. The test is read microscopically with an dark field microscope. 12Abed ElKader ElOttol

3. T. pallidum-Hemagglutination test (TP-HA): Red cells are treated to adsorb treponemal antigens on their surface. When mixed with serum containing antitreponemal antibodies, the cells become clumped. Clumping is considered as positive results. NB: VDRL and FTA-ABS tests can also be performed on spinal fluids. Antibodies do not reach the CSF from the blood stream but are probably formed in CNS in response to syphylitic infection. TREATMENT: Penicillin is the treatment of choice of syphilis 13Abed ElKader ElOttol

Diseases Related to Treponema: 1.Yaws: (Frambesia): is a tropical infection of the skin, bones and joints caused by T. pertenue. 2. Pinta is a human skin disease endemic to Mexico, Central America, and South America. It is caused by infection with a spirochete, Treponema carateum, which is morphologically and serologically indistinguishable from the organism that causes syphilis. Pinta initially presents as a raised papule, followed by a generalized eruption of flat, reddened areas, and is followed by the development of bluish coloration and a subsequent loss of pigmentation. Unlike syphilis, it is transmitted by nonsexual skin contact, often between children living in conditions of poor hygiene. The disease can be treated with penicillin, tetracycline, or chloramphenicol, and can be prevented through contact tracing by public health officials. 14Abed ElKader ElOttol

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16Abed ElKader ElOttol LEPTOSPIRA INTERROGANS

Abed ElKader ElOttol17 Causative agent of Leptospirosis : A zoonotic disease Habitat : Infected domestic live-stock مواشي & pets اليف. The organism settles in the kidney and excreted in urine. Mode of Infection Direct Contact with: Urine of infected animal Water & soil recently contaminated with infected urine The organism enters body through: Skin lesions Conjunctival mucus membrane Ingestion.

Abed ElKader ElOttol18 High-Risk Groups Farmers Sewer بالوعة workers LEPTOSPIROSIS Pathogenesis Incubation period : 1-2 weeks. Bacteremia : organisms multiply in liver, spleen, kidney, meninges, conjunctiva

Abed ElKader ElOttol19 Clinical Features Influenza-like followed by hepatitis & meningitis Weil’s Disease (severe leptospirosis) severe headache, pain in pack, prostration, jaundice Jaundice, hemorrhage, renal failure Lab Diagnosis Dark field Microscopy of blood & CSF Sero-diagnosis Leptospira can be cultured in Ellinghausen-McCullough- Johnson-Harris medium(EMJH), which is incubated at 28 to 30 °C. The median time to positivity is three weeks with a maximum of 3 months. Treatment & Prevention Penicillin Animal immunization Proper treatment and disposal of contaminated water

Abed ElKader ElOttol20 BORRELIA RECURRENTIS

Abed ElKader ElOttol21 Can be stained with Giemsa stain in blood film. Can grow in media containing serum & tissue extracts. High frequency of antigenic variation in major surface protein and is responsible for : Organism can escape immune system Relapses of disease Disease : Relapsing Fever Transmission : Tick-borne قراد Relapsing Fever From infected rodents to human Louse-borne قمل Relapsing Fever From infected human to human

Abed ElKader ElOttol22 Clinical Features Bacteremia : Infect various organs. A non-pruritic rash at bite site. Fever, rigors (shaking occurring during a high fever) and headache for weeks to months. Weeks to months later: Cardiac and neurological symptoms. Predominant arthritis. One of the causes of Pyrexia of Unknown Origin PUO. Lab Diagnosis Giemsa staining of blood smear. Detection of IgM OR rising titer of IgG. PCR. Treatment & Prevention Amoxycillin Louse, tick & rodent control Hygienic measures

Abed ElKader ElOttol23 End of Lecture