spirochetes

SPIROCHETESDr.T.V.Rao MD

Dr.T.V.Rao MD 1

SpirochetesSpirochetes -are elongated motile, flexible bacteria twisted spirally along the long axis are termed spirochetes

Contain – endoflegalla which are polar flagella wound along the helical protoplasmic cylinder and situated between the outer membrane and cell wall

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Order: SpirochaetalesFamily: Spirochaetaceae

Genus: Trepanoma Borrelia

Family: LeptospiraceaeGenus: Leptospira

Taxonomy

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Human pathogen

A. Genera Trepanoma

B. Borreilia

C. Leptospira

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How they appear

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What are Trepanoma

Trepos – Turn

Nema Meaning thread

Relatively short and slender

With fine spirals pointed and round ends

May be pathogenic or commensals in the mouth

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Genus Species Disease

Treponema pallidum ssp. pallidumpallidum ssp. endemicumpallidum ssp. pertenuecarateum

Syphilis Bejel YawsPinta

Borrelia burgdorferirecurrentisMany species

Lyme disease (borreliosis)

Epidemic relapsing feverEndemic relapsing fever

Leptospira interrogans Leptospirosis (Weil’s Disease)

Spirochaetales Associated Human Diseases

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Venereal Syphilis

Venereal Syphilis caused by T.pallidum Endemic syphilis T. pallidum

Yaws T.pertune

Pinta T.carateum

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Discovery“Everything” happened mostly in Germany from 1905 to 1910 !With a short life of 35 years, Fritz Schaudinn (1871-1906) and Paul E. Hoffmann (1868-1959) discovered Treponema pallidum in serum in 1905.

Paul Ehrlich, father of

immunochemistry and his assistent Hati.

Fritz Schaudinn Dr.T.V.Rao MD 9

SyphilisA. Named from poem

published by the Italian physician and poet Girolamo Fracastoro – shepherd from Hispaniola named Syphilis who angered Apollo and was given the disease as punishment

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Syphilis

"He who knows syphilis, knows

medicine"

Sir William Osler

Treponema pallidum

A. Described in 1905 by Schaudinn and Hoffman, Hamburg

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Trepanoma pallidumGreek words trepo “turning” & nema “head”

A. Morphology1. Motile, sluggish in viscous environments

2. Size: 5 to 20 μm in length & 0.09 to 0.5 μm in diameter, with tapered ends

3. Structure• Multilayer cytoplasmic membrane• Flagella-like fibrils• Cell wall• Outer sheath (outer cell envelope)• Capsule-like outer coat

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Treponema pallidum.

A. Spiral spirochete that is mobile of spirals varies from 4 to 14 Length 5 to 20 microns and very thin 0.1 to o.5 microns. Can be seen on fresh primary or secondary lesions by dark field microscopy or fluorescent antibody techniques

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General Overview of Spirochaetales

A. Gram-negative spirochetes

B. Spirochete from Greek for “coiled hair”

C. Extremely thin and can be very long

D. Tightly coiled helical cells with tapered ends

E. Motile by Periplasmic flagella (a.k.a., axial fibrils or endoflegalla)

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General Overview of Spirochaetales

A. Outer sheath encloses axial fibrils wrapped around protoplasmic cylinder

B. Axial fibrils originate from insertion pores at both poles of cell

C. May overlap at centre of cell in Treponema and Borrelia, but not in Leptospira

D. Differing numbers of endoflegalla according to genus & species

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Trepanoma palladium

B. Physiology– Difficult to culture

• Maintained in anaerobic medium with albumin, sodium bicarbonate, pyruvate, cysteine

• MicroaerophilicDr.T.V.Rao MD 17

Cross-Section of Spirochete

with Periplasmic

Flagella

NOTE: a.k.a., endoflegalla, axial fibrils or axial filaments.

(Outer sheath)

Cross section of Borrelia burgdorferi

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Staining with special stains

Staining by Giemsa and Fontana

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Antigenic structureA. The Antigens are complex

B. Infection with Treponema will induce 3 types of Antigens

C. Reagin Antibodies – STS D. Detected by Standard tests for

Syphilis

E. 1 Wasserman Test, 2 Kahn Test

F. VDRL Test Dr.T.V.Rao MD 20

Beef Heart Extracts - Antigen

Lipid Hapten – Cardiolipin Chemically Dipphostidyl glycerol

Cardiolipin present in the Trenonems ?

Or a product of tissue Damage ?

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Second Group Antigen T.pallidum

A.Present in T.pallidum and Non pathogenic cultivable treponemes

B.Reiter's Trenonems

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Third Antigen

Polysaccharide species specific

Positive only in sera of patients infected with pathogenic Treponema

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Dark field Microscopy

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Treponema cannot be cultivated in Culture Media

A. The inability to grow most pathogenic Treponema in vitro, coupled with the transitory nature of many of the lesions, makes diagnosis of Treponema infection impossible by routine bacteriological methods

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Cultivation of .. ?A. Although the Treponemes

are distantly related to Gram-negative bacteria, they do not stain by Gram's method, and modified staining procedures are used. Moreover, the pathogenic Treponemes cannot be cultivated in laboratory media and are maintained by subculture in susceptible animals.

Trepanoma pallidumD. Clinical Infection: Syphilis

1. Transmission• Usually through sexual contact from an infected

individual by invading intact mucous membranes or abraded skin

2. Pathogenesis• Disease of blood vessel & perivascular areas• Primary lesion due to inflammation at site of

inoculation• Secondary lesion due to inflammation of ectodermal

tissues• Tertiary lesion due to diffuse chronic inflammation to

organ systems

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Trepanoma pallidum

D. Clinical Infection: Syphilis3. Clinical Manifestations

i. Primary Disease• Chancre: single lesion, non-tender &

firm with a clean surface, raised border & reddish color

• Usually on the cervix, vaginal wall, anal canal

• Draining lymph nodes enlarged & non-tender

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Pathogenesis of T. pallidum Tissue destruction and lesions are primarily a

consequence of patient’s immune response Syphilis is a disease of blood vessels and of the

perivascular areas In spite of a vigorous host immune response the

organisms are capable of persisting for decades• Infection is neither fully controlled nor eradicated• In early stages, there is an inhibition of cell-mediated

immunity• Inhibition of CMI abates in late stages of disease, hence

late lesions tend to be localized

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Penetration: 1. T. pallidum enters the body via skin and

mucous membranes through abrasions during sexual contact

2. Also transmitted transplacentally

A. Dissemination: 1. Travels via the lymphatic system to regional

lymph nodes and then throughout the body via the blood stream

2. Invasion of the CNS can occur during any stage of syphilis

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Pathology

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A. The bacteria rapidly enter the lymphatic's, are widely disseminated via the bloodstream and may lodge in any organ. The exact infectious dose for man is not known, but in experimental animals fewer than ten organisms are sufficient to initiate infection.

Pathology

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Pathology

The bacteria multiply at the initial entry site forming a chancre, a lesion characteristic of primary syphilis, after an average incubation period of 3 weeks

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1. Primary2. Secondary3. Latent

i. Early latent ii. Late latent

4. Late or tertiary i. May involve any organ, but main parts are:

• Neurosyphilis• Cardiovascular syphilis• Late benign (gumma)

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STAGES OF SYPHILIS

Basic lesion of syphilis

The chancre is painless and most frequently on the external genitalia, but it may occur on the cervix, perianal area, in the mouth or anal canal.

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Stages of syphilisA. Untreated syphilis

may be a progressive disease with primary, secondary, latent and tertiary stages. T. pallidum enters tissues by penetration of intact mucosae or through abraded skin.

a) One or more painless chancres (indurated raise edges & clear bases) that erupt in the genitalia, anus, nipples, tonsils or eyelids.b) Starts as papule and then erodec) Disappear after three to six weeks even without treatment.d) Lymphadenopathy that is either unilateral or bilateral

Primary syphilis

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Trepanoma pallidumD. Clinical Infection: Syphilis

3. Clinical Manifestationsiii. Latent disease

a. Early latent (1st 4 years)• No signs & symptoms of active syphilis

but remain seroactive

b. Late latent (after 4 years)• If untreated, 60% continue to be

asymptomatic while 40% progress to tertiary stage

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PathologyA. The chancre is

painless and most frequently on the external genitalia, but it may occur on the cervix, peri-anal area, in the mouth or anal canal. Chancres usually occur singly, but in immunocompromised individuals,

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A. The chancre usually heals spontaneously within 3-6 weeks, and 2-12 weeks later the symptoms of secondary syphilis develop. These are highly variable and widespread but most commonly involve the skin where macular or pustular lesions develop, particularly on the trunk and extremities. The lesions of secondary syphilis are highly infectious.

Chancre

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A. Relapse of the lesions of secondary syphilis is common, and latent syphilis is classified as early (high likelihood of relapse) or late (recurrence unlikely). Individuals with late latent syphilis are not generally considered infectious, but may still transmit infection to the fetus during pregnancy and their blood may remain infectious.

Progress of Disease

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Trepanoma pallidum


i. Primary Disease• Chancre: single lesion, non-tender & firm

with a clean surface, raised border & reddish color

• Usually on the cervix, vaginal wall, anal canal

• Draining lymph nodes enlarged & non-tender Dr.T.V.Rao MD 41

A. Incubation period 9-90 days, usually ~21 days. B. Develops at site of contact/inoculation.C. Classically: single, painless, clean-based,

indurated ulcer, with firm, raised borders. Atypical presentations may occur.

D. Mostly anogenital, but may occur at any site (tongue, pharynx, lips, fingers, nipples, etc...)

E. Non-tender regional adenopathy F. Very infectious.G. May be darkfield positive but serologically negative. H. Untreated, heals in several weeks, leaving a faint scar.

PRIMARY SYPHILIS(The Chancre)

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Primary Syphilis

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Primary Syphilis- Penile Chancre

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Clinical Manifestations

Source: CDC/ NCHSTP/ Division of STD Prevention, STD Clinical Slides

Primary Syphilis

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Primary Syphilis - Chancre

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Primary Syphilis - Chancre

Secondary disease 2-10 weeks after primary lesion

Widely disseminated mucocutaneous rash

Secondary lesions of the skin and mucus membranes are highly contagious

Generalized immunological response

Pathogenesis of T. pallidum (cont.)

Secondary Syphilis

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Treponema pallidumD. Clinical Infection: Syphilis

3. Clinical Manifestationsiv. Tertiary Disease

a. Gummas (3-10 years after secondary disease)• Non-progressive, localized dermal lesions• Benign tertiary syphilis• Pronounced immunologic host reaction

b. Neurosyphilis (>5 years after primary disease)• Paralytic dementia, tabes dorsalis, amyotropic

lateral sclerosis, meningovascular syphilis, seizures, optic atrophy, gummatous changes of the cord Dr.T.V.Rao MD 49

Secondary SyphilisA. Secondary

syphilis at 6-8 weeks – diffuse symptoms:

1. Fever2. Headache3. Skin pustules

B. Usually disappears even without treatment

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Treponema pallidumD. Clinical Infection: Syphilis

3. Clinical Manifestations

v. Congenital Syphilisa. Transplacental infection of the developing fetus

b. Abortion occurs during 2nd trimester of pregnancy

c. Early symptoms: • Hepatosplenomegaly, jaundice, hemolytic

anemia, pneumonia, multiple long bone involvement, snuffles, skin lesions, testicular masses

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Treponema pallidum


v. Congenital Syphilis

d. Late symptoms:• Frontal bossae of Parrott, Short maxilla, high

palatal arch, Hutchinson’s triad (Hutchinson’s teeth, Interstitial keratitis, eighth-nerve deafness), saddle nose, mulberry molars, Higoumenakis sign, relative protruberance of mandible, rhagades, saber shin, scaphoid scapulas, Clutton’s joint)

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Secondary Syphilis

Secondary disease 2-10 weeks after primary lesion

Widely disseminated mucocutaneous rash

Secondary lesions of the skin and mucus membranes are highly contagious

Generalized immunological response


Secondary Syphilis

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Secondary Syphilis

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Secondary Syphilis

Secondary syphilis

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A. Affects 2/3 of untreated cases 1. Gummata: rubbery tumors2. Bone deformities3. Blindness4. Loss of coordination5. Paralysis6. Insanity

Tertiary Syphilis

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Following secondary disease, host enters latent period

•First 4 years = early latent

•Subsequent period = late latent

About 40% of late latent patients progress to late tertiary syphilitic disease


Latent Stage Syphilis

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Tertiary syphilis characterized by localized granulomatous dermal lesions (gummas) in which few organisms are present • Granulomas reflect containment by

the immunologic reaction of the host to chronic infection


Tertiary Syphilis

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Neurosyphilis A. Late Neurosyphilis develops in about 1/6

untreated cases, usually more than 5 years after initial infection

B. Central nervous system and spinal cord involvement

C. Dementia, seizures, wasting, etc.

D. Cardiovascular involvement appears 10-40 years after initial infection with resulting myocardial insufficiency and death

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A. Latent syphilisa) Reactive serologic testb) Asymptomatic until death

B. Late syphilisThree subtypes of Late syphilis

1. Late, benign syphilis *Develops between 1 to 10 years after the

infection *Presence of gumma

Latent Syphilis

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Mother to Child Transmission

Infection in utero may have serious consequences for the fetus. Rarely, syphilis has been acquired by transfusion of infected fresh human blood.

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Congenital syphilis results from trans placental infection

T. pallidum septicemia in the developing fetus and widespread dissemination

Abortion, neonatal mortality, and late mental or physical problems resulting from scars from the active disease and progression of the active disease state


Congenital Syphilis

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Treponema pallidum and Immunity

D. Clinical Infection: Syphilis4. Immunity

i. Syphilis has persistent infection for decades in spite vigorous host response due to:

• Dense coat (with fibronectin, transferrin, cerruloplasmin)• Evasion from PMN detection• Inhibition of cell-mediated immunity

ii. Relative but unreliable protection from reinfection in untreated patients

iii. Minor protection from reinfection in treated patients

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A. Passed from mother to fetus during pregnancy

1. Abnormally shaped teeth

2. Nasal septum collapses

3. Skeletal abnormalities

Congenital Syphilis

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A. 1. History and clinical examination.B. 2. Dark-field microscopy: special

technique use to demonstrate the spirochete as shiny motile spiral structures with a dark background.

C. The specimen includes oozing from the lesion or sometimes L.N. aspirate. It is usually positive in the primary and secondary stages and it is most useful in the primary stage when the serological tests are still negative.

DIAGNOSIS OF SYPHILIS

Diagnosis of syphilis

A. Direct detection of spirochetes :

Darkfield microscopy (motile bugs + experience + prompt examination)

Silver stain

B. Culture : not used

C. Serology: non-specific and specific tests

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Serologic TestsA. Reveal patients immune status not

whether they are currently infected B. Use lipoidal antigens rather than T.

pallidum or components of it; non-treponemal antigen tests

C. RPR; rapid plasma reaginD. VDRL; Venereal Disease Research

Laboratory

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Treponema pallidum

F. Laboratory diagnosis1. Serologic testing

i. Nontreponemal Tests (uses Cardiolipin-lecithin as antigen)

a. Complement-fixation tests (Wasserman & Kolmer test)

b. Flocculation tests (Venereal Disease Research Laboratory, (VDRL), Hinton & rapid reagin tests)

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Serologic Tests

A. Positive within 5 to 6 weeks after infection

B. Strongly positive in secondary phase

C. Strength of reaction is stated in dilutions

D. May become negative with treatment or over decades

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Treponema pallidum


ii. Treponemal Tests (uses syphilitic tissue as complement-fixing antigen)

b. Reiter Protein Complement Fixation• Antigen is an extract from nonvirulent

treponeme (Reiter strain)• Nonreactive in late stages of syphilis

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Non-treponemal tests

A. Antigen: cardiolipin (beef heart) + lecithin + cholesterol

B. Detect nonspecific antibody (Reagin): a mixture of IgM & IgG direct against some normal tissue antigens

C. VDRL (Venereal Disease Research Laboratory) test for serum and CSF samples

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A. Flocculation test, antigen consists of very fine particles that precipitate out in the presence of reagin.

B. Utilizes an antigen which consists of cardiolipin, cholesterol and lecithin.

1. Antigen very technique dependent.2. Must be made up fresh daily.

C. Serum must be heated to 56 C for 30 minutes to remove anti-complementary activity which may cause false positive, if serum is not tested within 4 hours must be reheated for 10 minutes.

D. Calibrated syringe utilized to dispense antigen must deliver 60 drops/mL +/- 2drops.

Venereal Disease Research Laboratory - VDRL

VDRL

A. Each preparation of antigen suspension should first be examined by testing with known positive or negative serum controls.

B. The antigen particles appear as short rod forms at magnification of about 100x. Aggregation of these particles into large or small clumps is interpreted as degrees of positivity

C. Reactive on left, non-reactive on rightDr.T.V.Rao MD 76

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A. General screening test, can be adapted to automation.

B. CANNOT be performed on CSF.C. Antigen

1. VDRL cardiolipin antigen is modified with choline chloride to make it more stable

2. attached to charcoal particles to allow macroscopic reading

3. antigen comes prepared and is very stable.

D. Serum or plasma may be used for testing, serum is not heated.

Rapid Plasma Reagin Test - RPR

Treponema pallidumF. Laboratory diagnosis

1. Serologic testing


a. Treponema Pallidum Immobilzation (TPI)• Reaginic antibody & complement immobilize a

suspension of living and motile treponemes maintained in rabbit testes & determined by darkfield microscopy

• Difficult, expensive, requires living organisms• Positive for nonvenereal treponematoses,

bejels, yaws & pintaDr.T.V.Rao MD 78

Treponema pallidum



b. Reiter Protein Complement Fixation• Antigen is an extract from nonvirulent

treponeme (Reiter strain)• Nonreactive in late stages of syphilis

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A. A. Reiter protein complement fixation test.

B. B. Fluorescent Treponemal antibody/absorption test, FTA/ABS. the most specific and most sensitive .

C. C. Treponema pallidum haemagglutination test- TPHA- D. Treponema pallidum immobilization test- TPI

Specific serological tests of syphilis

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Treponema pallidum haemagglutination (TPHA)

A. Adapted to micro techniques (MHA-TP)

B. Tanned sheep RBCs are coated with T. pallidum antigen from Nichol’s strain.

C. Agglutination of the RBCs is a positive result.

Specific serological tests of syphilis

A. A. Reiter protein complement fixation test.

B. B. Fluorescent Treponemal antibody/absorption test, FTA/ABS. the most specific and most sensitive .

C. C. Treponema pallidum Haemagglutination test- TPHA- D. Treponema pallidum immobilization test- TPI Dr.T.V.Rao MD 82

Treponema pallidum



c. Fluorescent Antibody Tests / Fluorescent Treponemal Antibody Absorption (FTA-ABS) Test• Uses lyophilized Nichols strain organisms as

antigen mixed with antitreponemal antibody (from test serum) in a slide flourescein isothiocyanate-labeled antihuman Ig –> presence of antibody determined by darkfield microscopy

• Used to diagnosed congenital syphilis & late stage syphilis, confirmation of nontreponemal tests

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A. Diluted, heat inactivated serum added to Reiter’s strain of T. pallidum to remove cross reactivity due to other Treponemes.

B. Slides are coated with Nichol’s strain of T. pallidum and add absorbed patient serum.

C. Slides are washed, and incubated with antibody bound to a fluorescent tag.

D. After washing the slides are examined for fluorescence.

E. Requires experienced personnel to read.F. Highly sensitive and specific, but time

consuming to perform.

Fluorescent Treponemal Antibody Absorption Test (FTA-ABS)

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Positive FTA Test for Syphilis Viewed with a Fluorescent Microscope

Serologic TestsA. To improve sensitivity and specificity tests

using a specific treponemal antigen devised

B. MHA-TP: microhemagglutination assay for T. pallidum

C. FTA-ABS: fluorescent treponemal antibody absorption test

D. All positive nontreponemal test results should be confirmed with a specific treponemal test

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Treponema pallidumF. Laboratory diagnosis

1. Serologic testingii. Treponemal Tests (uses syphilitic

tissue as complement-fixing antigen)

d. Haemagglutination Tests

a. Microhemagglutination assay –T. pallidum (MHA-TP)

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Every Pregnant women Needs Screening

Biologic False-Positive Test Results

A. Positive STS in persons with no history or clinical evidence of syphilis

B. Acute BFP: those that revert to negative in less than 6 months

C. Chronic BFP: persist > 6 months

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BFP Test Results in Syphilis

A. Acute BFP

B. Vaccinations

C. Infections

D. pregnancy

A. Chronic BFP

B. Connective tissue disease (SLE)

C. Liver disease

D. Blood transfusions

E. IVDA

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Advantage of VDRL:

• cheap, easy to perform

• quantitative, screen test

• monitor disease course

• trace theraputic effect, become “-” in

6-18 m after effective treatment.

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A. Late syphilis:B. benzathine penicillin 2.4 million units intramuscularly

weekly for 3 weeks.C. procaine penicillin 1.2 million units intramuscularly

daily for 21 daysD. Tetracycline or erythromycin 500 mg 4 times a day –

or doxycycline 100 mg x2- by mouth for 30 days

E. Jarrisch-Herxheimer reaction

F. Follow-up

Treatment of Late Syphilis

Prevention & Treatment of Syphilis

Penicillin remains drug of choice• WHO monitors treatment recommendations• 7-10 days continuously for early stage• At least 21 days continuously beyond the early

stage Prevention with barrier methods (e.g.,

condoms) Prophylactic treatment of contacts

identified through epidemiological tracing

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Treponema pallidumG. Treatment & Prevention

1. Antibiotic treatment • DOC: Penicillin (complete recovery for stage

I &II)• streptomycin, tetracycline, erythromycin

(poor passage to fetal circulation)

2. Treatment of case contacts

3. Barrier methods (condom); “safe sex”

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Other Related to Treponemes

A. Related Treponemes cause the non-venereal treponematoses bejel, or endemic syphilis (T. pallidum endemicum), yaws (T. pallidum pertenue), and pinta (T. carateum).

Treponema pallidum

G. Other treponemal diseases1. Yaws (Frambesia) -Treponema pertenue

• Resembles syphilis• Acquired in childhood other than sexual contact• Mother yaw (or framboise), a painless erythematous

papule occurs a month after primary infection• Secondary lesion resemble primary lesion occurs 1-3

months after• Tertiary lesions involve the skin & bones, crab yaws• DOC: Penicillin

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Treponema pallidumG. Other treponemal diseases

2. Pinta - Treponema carateum• Acquired by person-to-person contact &

rarely by sexual contact• Primary & secondary lesions are flat,

erythematous & non-ulcerating; healing first becomes hyper pigmented and later depigmented scarring; occurs in hand, feet & scalp

• Tertiary lesions are uncommon• DOC: Penicillin

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Treponema pallidum

G. Other treponemal diseases3. Bejel - Treponema pallidum

variant• Endemic syphilis• Acquired by direct contact during

childhood• Similar to syphilis• DOC: PenicillinDr.T.V.Rao MD 98

Programme created by Dr.T.V.Rao MD for Medical and

Health Care Workers in the Developing World

Email

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spirochetes

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