SPIROCHETES

Dr.T.V.Rao MD

Dr.T.V.Rao MD

Spirochetes
Spirochetes -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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Taxonomy Order: Spirochaetales

Family: Spirochaetaceae Genus: Trepanoma Borrelia Family: Leptospiraceae Genus: Leptospira
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Human pathogen
A. Genera Trepanoma

B. C.

Borreilia 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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Spirochaetales Associated Human Diseases

Genus Species Syphilis Bejel Yaws Pinta Lyme disease (borreliosis) Epidemic relapsing fever Endemic relapsing fever Leptospirosis (Weils Disease)
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Disease

Treponema pallidum ssp. pallidum pallidum ssp. endemicum pallidum ssp. pertenue carateum Borrelia burgdorferi recurrentis Many species interrogans

Leptospira

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 Dr.T.V.Rao MD Schaudinn

Syphilis
A. 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 Dr.T.V.Rao MD

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Syphilis
"He who knows syphilis, knows medicine" Sir William Osler
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Treponema pallidum
A. Described in 1905 by Schaudinn and Hoffman, Hamburg
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Trepanoma pallidum
Greek words trepo turning & nema head

A.
1. 2. 3.

Morphology
Motile, sluggish in viscous environments Size: 5 to 20 m in length & 0.09 to 0.5 m in diameter, with tapered ends 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 Microaerophilic
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Cross section of Borrelia burgdorferi

Cross-Section of Spirochete with Periplasmic Flagella

NOTE: a.k.a., endoflegalla, axial fibrils or axial filaments.
(Outer sheath)
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Staining with special stains

Staining by Giemsa and Fontana

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Antigenic structure
A. 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

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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.
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Trepanoma pallidum
D. 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: Syphilis
3. 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 & nontender
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Pathogenesis of T. pallidum
Tissue destruction and lesions are primarily a consequence of patients 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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Pathology
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
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.
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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 Dr.T.V.Rao MD

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STAGES OF SYPHILIS
1. Primary 2. Secondary 3. 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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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 syphilis
A. 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.
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Primary syphilis
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 erode c) Disappear after three to six weeks even without treatment. d) Lymphadenopathy that is either unilateral or bilateral
a)
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Trepanoma pallidum
D. Clinical Infection: Syphilis
3. Clinical Manifestations
iii. 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 tertiaryDr.T.V.Rao MD stage

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Pathology
A. 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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Chancre
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.
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Progress of Disease
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. Dr.T.V.Rao MD 40

Trepanoma pallidum
D. Clinical Infection: Syphilis
3. 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 & nontender Dr.T.V.Rao MD 41

PRIMARY SYPHILIS (The Chancre)

A. B. C.
D. E. F. G.
H.

Incubation period 9-90 days, usually ~21 days. Develops at site of contact/inoculation. Classically: single, painless, clean-based, indurated ulcer, with firm, raised borders. Atypical presentations may occur. Mostly anogenital, but may occur at any site (tongue, pharynx, lips, fingers, nipples, etc...) Non-tender regional adenopathy Very infectious. May be darkfield positive but serologically negative.
Untreated, heals in several weeks, leaving a faint scar.
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Primary Syphilis

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Clinical Manifestations

Primary Syphilis- Penile Chancre

Dr.T.V.Rao MD Source: CDC/ NCHSTP/ Division of STD Prevention, STD Clinical Slides

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Primary Syphilis

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Primary Syphilis - Chancre

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Primary Syphilis - Chancre

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Pathogenesis of T. pallidum (cont.) 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
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Treponema pallidum
D. Clinical Infection: Syphilis
3. Clinical Manifestations
iv. 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 Dr.T.V.Rao MD 49 of the cord

Secondary Syphilis
A. Secondary syphilis at 6-8 weeks diffuse symptoms:
1. 2. 3. Fever Headache Skin pustules

B. Usually disappears even without treatment

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Treponema pallidum
D. Clinical Infection: Syphilis
3. Clinical Manifestations v. Congenital Syphilis
a. b. c. Transplacental infection of the developing fetus Abortion occurs during 2nd trimester of pregnancy Early symptoms: Hepatosplenomegaly, jaundice, hemolytic anemia, pneumonia, multiple long bone involvement, snuffles, skin lesions, testicular masses

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Treponema pallidum
D. Clinical Infection: Syphilis
3.
v.

Clinical Manifestations
Congenital Syphilis

d. Late symptoms:
Frontal bossae of Parrott, Short maxilla, high palatal arch, Hutchinsons triad (Hutchinsons teeth, Interstitial keratitis, eighth-nerve deafness), saddle nose, mulberry molars, Higoumenakis sign, relative protruberance of mandible, rhagades, saber shin, scaphoid scapulas, Cluttons joint)
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Secondary Syphilis

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Pathogenesis of T. pallidum (cont.) 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
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Secondary Syphilis

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Secondary Syphilis

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Secondary syphilis

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Tertiary Syphilis
A. Affects 2/3 of untreated cases
1. 2. 3. 4. 5. 6. Gummata: rubbery tumors Bone deformities Blindness Loss of coordination Paralysis Insanity
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Pathogenesis of T. pallidum (cont.) Latent Stage Syphilis

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
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Pathogenesis of T. pallidum (cont.) Tertiary Syphilis 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
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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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Latent Syphilis
A. Latent syphilis
a) Reactive serologic test b) Asymptomatic until death

A. Late syphilis
Three subtypes of Late syphilis

1. Late, benign syphilis

*Develops between 1 to 10 years after the infection *Presence of gumma
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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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Pathogenesis of T. pallidum (cont.) Congenital Syphilis

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
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Treponema pallidum and Immunity

D. Clinical Infection: Syphilis
4.
i.

Immunity
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. iii.

Relative but unreliable protection from reinfection in untreated patients Minor protection from reinfection in treated patients

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Congenital Syphilis
A. Passed from mother to fetus during pregnancy

1. Abnormally shaped teeth 2. Nasal septum collapses 3. Skeletal abnormalities

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DIAGNOSIS OF SYPHILIS
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.
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Diagnosis of syphilis
A. Direct detection of spirochetes :
Darkfield microscopy (motile bugs + experience + prompt examination) Silver stain A. Culture : not used B. Serology: non-specific and specific tests Dr.T.V.Rao MD

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Serologic Tests
A. Reveal patients immune status not whether they are currently infected B. Use lipoidal antigens rather than T. pallidum or components of it; nontreponemal antigen tests C. RPR; rapid plasma reagin D. VDRL; Venereal Disease Research Laboratory
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Treponema pallidum
F. Laboratory diagnosis
1. 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), Dr.T.V.Rao MD & rapid reagin tests) Hinton 71

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
F. Laboratory diagnosis
1. Serologic testing
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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Venereal Disease Research Laboratory - VDRL

A.
B.

Flocculation test, antigen consists of very fine particles that precipitate out in the presence of reagin. Utilizes an antigen which consists of cardiolipin, cholesterol and lecithin.
1. 2. Antigen very technique dependent. Must be made up fresh daily.

C.

D.

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

A. B.

C.

Each preparation of antigen suspension should first be examined by testing with known positive or negative serum controls. 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 Reactive on left, non-reactive on right
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Rapid Plasma Reagin Test - RPR

A. General screening test, can be adapted to automation. B. CANNOT be performed on CSF. C. Antigen
1.
2. 3.

VDRL cardiolipin antigen is modified with choline chloride to make it more stable attached to charcoal particles to allow macroscopic reading antigen comes prepared and is very stable.

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

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Treponema pallidum
F.
1.

Laboratory diagnosis
Serologic testing

ii.

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

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 & pinta
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Treponema pallidum
F. Laboratory diagnosis
1. Serologic testing
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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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 testDr.T.V.Rao MD 80 TPI

Treponema pallidum haemagglutination (TPHA)

A. Adapted to micro techniques (MHATP) B. Tanned sheep RBCs are coated with T. pallidum antigen from Nichols strain. C. Agglutination of the RBCs is a positive result.
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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 Dr.T.V.Rao MD 82 test- TPI

Treponema pallidum
F.
1.
ii.

Laboratory diagnosis
Serologic testing
Treponemal Tests (uses syphilitic tissue as complement-fixing antigen)
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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Fluorescent Treponemal Antibody Absorption Test (FTA-ABS)

A. Diluted, heat inactivated serum added to Reiters strain of T. pallidum to remove cross reactivity due to other Treponemes. B. Slides are coated with Nichols 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 Dr.T.V.Rao MD and specific, but time 84 consuming to perform.

Positive FTA Test for Syphilis Viewed with a Fluorescent Microscope

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Serologic Tests
A. 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 pallidum
F. Laboratory diagnosis

1. Serologic testing
ii. 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

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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. B. C. D. Acute BFP Vaccinations Infections 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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Treatment of Late Syphilis

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 days D. 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
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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 pallidum
G. 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).
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Treponema pallidum
G. Other treponemal diseases
1.

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 pallidum
G. 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 Dr.T.V.Rao MD 97

Treponema pallidum
G. Other treponemal diseases
3. Bejel - Treponema pallidum variant
Endemic syphilis Acquired by direct contact during childhood Similar to syphilis Dr.T.V.Rao MD 98 DOC: Penicillin

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