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SPIROCHAETES Dr.Lancy.J Associate Professor 6/29/2011 1

Spirochaetes :

Spirochaetes Elongated Motile Flexible helical bacteria t wisted spirally along the long axis Spira = coil Chaite = hair Structurally more complex They are free living organisms found in water and sewage. Presence of endoflagella situated between outer membrane and cell wall- responsible for motility 6/29/2011 2

Human pathogens:

Human pathogens 3 genera Treponema Trepos = to turn Nema = thread Borrelia Leptospira 6/29/2011 3

Treponema :

Treponema Treponema pallidum Syphilis Treponema pertenue Yaws Treponema carateum Pinta Treponema endemicum Endemic syphilis 6/29/2011 4

Treponema pallidum:

Treponema pallidum Causative agent of Syphilis STD Thin delicate spirochete with tapering ends 10 µm long 0.1-0.2 µm wide 10 regular spirals – sharp angular at regular intervals of 1 µm Actively motile, exhibiting rotation around long axis – backward and forward movement with flexion of whole body 6/29/2011 5

Staining :

Staining not s tained by ordinary stains Giemsa stain – stains light rose red Silver impregnation method Negative stain – Indian Ink Morphology and motility can be seen under dark ground microscope or phase contrast microscope 6/29/2011 6

Structure :

Structure Cell wall – peptidoglycan which gives the cell rigidity and shape Trilaminar cytoplasmic membrane Outer membrane – lipid Endoflagella – 3-4 in number wind around the axis of the cell in the space between cell wall and outer membrane 6/29/2011 7

Culture :

Culture Do not grow in artificial media Limited growth in tissue culture Virulent strains have been maintained by serial testicular passage in rabbit – Nichol’s strain Non pathogenic treponemes show morphological and antigenic similarities with treponema pallidum – REITER STRAIN 6/29/2011 8

Culture …..:

Culture ….. Reiter treponemes grow well in Thioglycollate medium containing serum Reiter treponemes have been widely used as the antigen in group specific treponemal tests for the diagnosis of Syphilis 6/29/2011 9

Resistance :

Resistance T.pallidum is a very delicate organism Inactivated by drying/heat 41-42 0 C/hr Susceptibility to heat was the basis of the ‘ fever therapy ’ for syphilis Killed in 1-3 days at 0-4 0 C. So that transfusion syphilis can be prevented by storing blood for at least 4 days Inactivated by contact with oxygen, soap, arsenicals, mercurials , bismuth, common antiseptics and antibiotics 6/29/2011 10

Antigenic structure:

Antigenic structure Cardiolipin antigen-common antigen for treponemes Group specific antigen-Found in T.pallidum as well as in non pathogenic cultivable Reiter treponemes Species specific polysaccharide antigen- antigen specific for T reponema pallidum 6/29/2011 11

Cardiolipin antigen :

Cardiolipin antigen Chemically a diphosphatidyl glycerol This lipid has been detected in T.pallidum Used as antigen in the standard tests for syphilis [STS] or nonspecific tests for syphilis Wassermann test Kahn test VDRL test 6/29/2011 12


REAGIN ANTIBODY The antibody that reacts with the Cardiolipin antigen – Reagin antibody It is not known whether the Reagin antibody is induced by cardiolipin that is present in the spirochete or released from damaged host tissues Reagin antibody detectable 7-10 days after primary chancre 6/29/2011 13

Syphilis :

Syphilis Sexually transmitted disease Infective dose - as few as 60 treponemes Incubation period - 10- 90 days 3 stages Primary Secondary Tertiary 6/29/2011 14

Primary Syphilis:

Primary Syphilis Presence of Chancre at the site of entry of spirochete Chancre is a painless, relatively avascular , circumscribed, indurate, superficially ulcerated lesion Common sites – genitalia, mouth, nipples, Chancre is covered by a thick, glairy exudate , very rich in spirochetes 6/29/2011 15


Chancre 6/29/2011 16

Primary Syphilis…..:

Primary Syphilis….. Regional lymph nodes are swollen, discrete, rubbery and non tender It heals in 10-40 days even with out treatment leaving a thin scar Even before the chancre appears, the spirochetes spread from the site of entry into the lymph and blood steam Patient may be infective Persistent and multiple chancres can be seen in HIV patients 6/29/2011 17

Secondary Syphilis:

Secondary Syphilis Sets in 3 months after primary lesion heals During this interval patient is asymptomatic Secondary lesions are due to wide spread multiplication of the spirochetes and their dissemination through blood 6/29/2011 18

Secondary Syphilis…..:

Secondary Syphilis….. Spirochetes are abundant in the lesions Patient is most infectious Roseolar or papular skin rashes , mucus patches in the oropharynx Condylomata at muco-cutaneous junctions are the characteristic lesions 6/29/2011 19

Roseolar skin rashes:

Roseolar skin rashes 6/29/2011 20

Secondary Syphilis……:

Secondary Syphilis…… There may be ophthalmic, osseous, and meningeal involvement Secondary lesions are highly variable in distribution, intensity and duration. But they usually undergo spontaneous healing in some instance 4-5 years 6/29/2011 21

Latent Syphilis:

Latent Syphilis After the secondary lesions disappear, three is a period of quiescence known as latent syphilis Diagnosis during this period is possible only by serological tests 6/29/2011 22

Tertiary Syphilis:

Tertiary Syphilis After the period of latent syphilis in many cases natural cure Others manifestations of tertiary syphilis CVS syphilitic aneurysm Chronic granulomata gummata Meningo -vascular meningitis Neurological tabes dorsalis and general paralysis of insane [GPI] 6/29/2011 23

Natural evolution of non venereal syphilis :

Natural evolution of non venereal syphilis Syphilis acquired non venereally as occupational in doctors and nurses Natural evolution as in venereal syphilis except the primary chancer is extra genital usually on fingers 6/29/2011 24

Transfusion syphilis :

Transfusion syphilis Syphilis acquired by blood transfusion Primary chancer does not occur Can be prevented by storing of blood 0 to 4 0 C at least for 4 days before tranfusion 6/29/2011 25

Congenital syphilis:

Congenital syphilis When infection is transmitted from mother to foetus trans- placentally Can occur at any stage of pregnancy A woman with early syphilis can infect her foetus much more commonly -75to 95% The lesion of congenital syphilis usually develops only after the 4 th month of gestation – at the time of foetal immune competence begins 6/29/2011 26

Slide 27:

Congenital syphilis can be prevented if the mother is given adequate treatment before 4 th month of pregnancy The obstetric history of an un treated syphilitic woman is typically one of abortions and still birth followed by live birth of infants with stigma of syphilis and finally of healthy infants 6/29/2011 27

Hunterian chancer :

Hunterian chancer Primary lesion that appears in syphilis It is painless relatively avascular circumscribed indurated superficially ulcerated lesion Named after John hunter who produced the lesion on himself experimentally and described the evolution of disease 6/29/2011 28

Laboratory diagnosis of syphilis :

Laboratory diagnosis of syphilis Microscopy Detection of antibodies in serum Test for antibodies reacting with cardiolipin antigen VDRL( Venereal D isease R esearch L aboratory test ) RPR ( Rapid Plasma R eagin Test) Test for antibodies reacting with group specific antigen RPCF (REITER PROTIEN COMPLIMENT FIXATION TEST) Not in use now Test for antibodies reacting with species specific antibodies TPI( T.pallidum Immobilisation test) TPIA ( T.pallidum Immune adherence test) TPA ( T.pallidum A gglutination test ) TPHA ( T.pallidum H aemagglutination assay ) 6/29/2011 29


VDRL Developed in New york Test is done on VDRL slide – To the Inactivated serum [heating serum at 55 0 C]taken in dilutions 1 drop of cardiolipin antigen is added Mix well in VDRL rotator [ 180 rpm/4mts ] Visible clumps/floccules appear on the slide if the patient serum contain antibody Seen under low power microscope Serial dilution to determine Ab titer in positive cases 6/29/2011 30


VDRL 6/29/2011 31 VDRL slide VDRL rotator

Biological false positive reaction :

Biological false positive reaction As cardiolipin antigen is present in T.pallidum and in mammalian tissues reagin antibodies may be induced by treponemal or host tissue antigens. This account for the BFP reactions 6/29/2011 32

Major disadvantage of STS:

Major disadvantage of STS BIOLOGICAL FALSE POSITIVE TESTS Positive – cardiolipin tests Negative results in specific Treponemal tests Absence of past/present Treponemal Infections Not caused by technical faults BFP antibody is usually IgM BFP occurs in about 1% of normal sera 6/29/2011 33


BIOLOGICAL FALSE POSITIVE TESTS Acute BFP Acute infections Injuries Inflammatory conditions Chronic BFP SLE & other collagen disease Leprosy Malaria Relapsing fever Infectious mononucleosis Hepatitis Tropical eosinophilia 6/29/2011 34

RPR(Rapid plasma reagin test):

RPR(Rapid plasma reagin test) Almost similar to VDRL Finely divided carbon particles added to cardiolipin antigen unheated serum/plasma can be used A finger prick sample of blood is sufficient No need for microscope for reading commercially available kit It cannot be used with CSF 6/29/2011 35


TRUST Toludine red unheated serum test Modified RPR test Commercially available kit Toludine red particles used instead of carbon particles Automated RPR and automated VDRL- Elisa have been developed Used for large scale test 6/29/2011 36

T.pallidum Immobilization Test:

T.pallidum Immobilization Test Test serum + compliment+ complex medium incubate anaerobically If antibodies + treponema are immobilized when examined under DGI microscope 50% or more immobilized positive 20 or less immobilized negative In between inconclusive Most sensitive & specific but dangerous 6/29/2011 37

Treponema pallidum haemagglutination assay:

Treponema pallidum haemagglutination assay Test serum is absorbed with a diluents containing components of Reiter treponemes, rabbit testis and sheep erythrocytes Tanned erythrocytes sensitized with a sonicated extract of T.pallidum Haemagglutination occur if the patients serum contains antibodies agaiinst T. pallidum Initial dilution 1: 80 5120 or more is common in secondary stage TPHA Most useful in Neurosyphilis once positive life long positive 6/29/2011 38


FTA - ABS Fluorescent Treponemal antibody- absorption test FTA-ABS Most specific and sensitive Serum first reacted with sorbent to absorb out non specific T . Antibodies Absorbed serum applied to a slide covered with killed TP suspension as the Ag Fluorescin conjugated anti human Ig serum applied Slides examined under UV microscope fitted with dark ground condenser 6/29/2011 39

Slide 40:

Antigen Patient’s serum with Ab Fluorescent anti human Ig antibody fluorescent treponemes 6/29/2011 40 Positive reaction Pt’s serum with out Ab Fluorescent antihuman Ig No fluorescent treponemes Negative reaction Antigen

Slide 41:

Stage VDRL FTA-ABS TPHA PRIMARY 70-80% 85-100% 65-85% SECONDARY 100% 100% 100% TERTIARY 60-70% 95-100% 95-100% 6/29/2011 41 Comparison of non specific and specific tests during different stages of syphilis

Treatment :

Treatment Benzathine penicillin G Single injection 2.4 million units in early cases Repeated wkly x 3 wks in late cases In patients allergic to penicillin Erythromycin Tetracycline Ceftriaxone 6/29/2011 42

Side effects:

Side effects Jarisch – Herxheimer reaction Fever, malaise, exacerbation of symptoms Due to liberation of toxic products from the massive destruction of treponemes or due to hypersensitivity 6/29/2011 43

Immunity :

Immunity Re-infections do not appear to occur in a person already having active infection Premunition immunity / Infection immunity A patient become susceptible to re-infection only when his original infection is cured 6/29/2011 44

Syphilis and HIV:

Syphilis and HIV Concurrent infection with Syphilis and HIV is common and lead to earlier evolution of Neurosyphilis 6/29/2011 45

Slide 46:

6/29/2011 46