Published on February 16, 2014
Spirochetes Treponema pallidum
Spirochaetes • Elongated Motile Flexible helical bacteria • twisted 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
Human pathogens • 3 genera • Treponema – Trepos = to turn – Nema = thread • Borrelia • Leptospira
Treponema • • • • Treponema pallidum Syphilis Treponema pertenue Yaws Treponema carateum Pinta Treponema endemicum Endemic syphilis
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
Staining • not stained 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
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
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 • 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
Resistance • T.pallidum is a very delicate organism • Inactivated by drying/heat 41-420C/hr • Susceptibility to heat was the basis of the ‘fever therapy’ for syphilis • Killed in 1-3 days at 0-40 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
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 Treponema pallidum
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
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
Syphilis • Sexually transmitted disease • Infective dose - as few as 60 treponemes • Incubation period 10- 90 days 3 stages – Primary – Secondary – Tertiary
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
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
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
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
Roseolar skin rashes
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
Latent Syphilis • After the secondary lesions disappear, there is a period of quiescence known as latent syphilis • Diagnosis during this period is possible only by serological tests
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]
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
Transfusion syphilis • Syphilis acquired by blood transfusion • Primary chancer does not occur • Can be prevented by storing of blood 0 to 40C at least for 4 days before tranfusion
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
• Congenital syphilis can be prevented if the mother is given adequate treatment before 4th 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
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
Laboratory diagnosis of syphilis 1. Microscopy 2. Detection of antibodies in serum a) b) I. Test for antibodies reacting with cardiolipin antigen I. II. VDRL( Venereal Disease Research Laboratory test ) RPR ( Rapid Plasma Reagin Test) I. RPCF (REITER PROTIEN COMPLIMENT FIXATION TEST) Not in use now Test for antibodies reacting with group specific antigen Test for antibodies reacting with species specific antibodies I. II. III. IV. TPI( T.pallidum Immobilisation test) TPIA (T.pallidum Immune adherence test) TPA ( T.pallidum Agglutination test ) TPHA ( T.pallidum Haemagglutination assay )
VDRL • Developed in New york • Test is done on VDRL slide – • To the Inactivated serum [heating serum at 550C]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
VDRL VDRL slide VDRL rotator
RPR(Rapid plasma reagin test) • Almost similar to VDRL • Finely divided carbon particles added to cardiolipin antigen 1. 2. 3. 4. 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
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
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
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
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
• Described by Weil & Stimson • Leptospires are actively mobile, delicate spirochetes possessing large number of closely wound spirals & characteristic hooked ends. • Leptospires – Saprophytic species – Pathogenic species • Pathogenic species are grouped under leptospira interrogans & saprophytic species grouped under leptospira biflexa • Leptospira interrogans classified into serogroups based on surface antigens.
Leptospira Vector Disease Icterohemorrhagica Rat Weil’s disease Canicola Dog Canicola fever Grippothyposa Field mice Swamp or marsh fever Pomona Pig Swine heard’s fever Hebdomadis Field mice Seven day fever Fortbragg Pretibial fever Pyrogenes Pig Febrile spirochetosis Bataviae Rat Indonesian weil’s disease Hardjo Cattle Dairy farm fever
Morphology • • • • • Delicate flexible helical rods 6-20μm long & 0.1 μm thick Actively motile Aerobic & microaerophillic Stain poorly with aniline dyes Numerous coils close to each other with a distinct hooked ends resemling umbrella handle. • Can be stained with with giemsa’s stain • Better visuvalized using silver impregnation method
Culture • Optimum temperature 25-30°C • pH 7.2 – 7.5 • Generation time – In culture 12-16hours – In animal inoculation 4-8hours • Media used EMJH (Ellinghausen, Mc Collough, Johnson, Harris) • Other media used enriched media with rabbit serum – Korthof’s – Stuarts – Fletcher’s medium
• Animal inoculation • Chorio-allantoic membrane of chick embryo • Demonstration in blood of allantoic vessels 4-5days after inoculation • Intra peritoneal inoculation of guinea pigs • Culture of heart blood 10 mins later
Clinical features • • • • Evidence of inapparent infection in 15-40% Incubation period is 2-20days 90% of symptomatic patients develop anicteric leptospirosis 10% of symptomatic patients develop weil’s disease ( icteric leptospirosis) • Anicteric Leptospirosis – Flu like syndrome – Classically severe muscle pain in calves, back & abdomen – Intense headache in frontal or reterobulbar region – Pulmonary symptoms – cough & chest pain – Eye – conjunctival congestion – Others pharyngeal congestion, rash, hepatomegaly, spleenomegaly, mild jaundice.
• • • Jaundice – severe – Gives an orange cast to the skin – Not associated with hepatic necrosis – Hepatomegaly – Spleenomegaly in 20 % Renal dysfunction – Seen during 2nd week of illness. – Mainly present with acute tubular necrosis with oliguria or anuria. – Renal function is completely reversible Hemorrhagic diathesis – Epistaxis , Petechiae, Purpura, Ecchymosis – Rarely severe G.I bleed, adrenal or subarachanoid hemorrhage – Fatal consequences due to Rhabdomyolysis, Hemolysis, Myocarditis, Pericarditis, Congestive heart failure, Cardiogenic shock, ARDS, Necrotizing pancreatirtis& Multi organ failure.
Lab Diagnosis Demonstration of spirochetes in blood in early stages Demonstration of spirochetes in urine in later stages Isolation in culture Animal inoculation Serological tests.
• Examination of blood – Useful in the first one week of disease & before antibiotic administration. – Dark field microscopy & Immunofluroscence is practically difficult. – Culture 3-4drops of blood inoculated in EMJH media @ 37°C for 2days and left thereafter at room temperature in dark for 2 weeks. • Samples from culture examined every 3 days – Animal inoculation • Intraperitoneal inoculation into young guinea pigs – Virulent strain – death in 8-12 days. – Less virulent strain
• Urine examination – Appear in urine from 2 week and then intermittently upto 4-6 weeks. – Urine sample should be examined immediately to prevent lysis by acidic urine – Centrifuges urine deposit examined by dark ground illumination
Treatment & Prophylaxis • • Penicillin or tetracycline Jarisch herxheimer like reaction • • • Rodent control Disinfection of water Protective clothing • Vaccination is serotype specific success rate is unpredictable
borrelia • Borrelias are a species of spirochetes of which many are Commensals of buccal & genital mucousa. • • • • • • Relapsing fever – borrelia recurrentis Lyme’s disease – borrelia burgdorferi Vincent’s angina- borrelia vincentii Neuroborreliosis – borellia garinii Arthritis & chronic skin lesions – borellia afzelii Normal commensal – borrelia buccalis.
Morphology • Gram negative spirochete, they are large, motile with 10 axial filaments attached at both ends of the organism. • They are refractile spirochetes with irregular wide open coils. • They are usually 10-30μm in length and 0.30.7μm wide.
Name East African relapsing fever Asian relapsing fever Causative organism B.duttonii Vector Ornithodoros moubata b.persica Ornithodoros tholozanii Relapsing fever in USA b.turicatae o.turicatae Lyme’s disease b.parkeri b.hermsii b.burgdorferi o.parkeri o.hermsii Ixodes dammcnii – USA Ixodes pacificus – USA Ixodes ricinus – Europe & switz Vincent’s angina b.vincentii
How to grow? • Barbour Stoenner Kellymedium ( BSK II) • Growth best at 34°C • Spectra of microaerophillic spirochetes( B.Burgdorferi) to anerobic spirochetes (B.vincentii) • Borrelia duttoni can be maintained in ticks for a long period in a sand box kept at room temperature. • Ticks feed on new born mice once a year. ticks remain infective and borrelias retain their virulence for many years. • Borrelias have been preserved in ticks & lice deep frozen at -76°C
Relapsing fever • • • • • • • • • • • Tick borne infection Soft ticks can survive as long as 10 years with occasional blood meal. Incubation period 7-10days Clinical features – patients present with headache, myalgia, chills, nausea, arthralgia, vomiting, abdominal pain, confusion, dry cough, eye pain, diarrhoea, dizziness, photophobia, neck pain. Arthralgia is severe involving small & large joints, but no evidence of evidence of arthritis. Fever is generally >104°C and irregular pattern. Tachycardia +, some patients may have symptoms of meningeal irritation. Eye – congestion, photophobia, icterus Skin & mucous membrane – signs of dehydration, petechiae over trunk, extremities & mucous membrane. R/S - non productive cough CVS – high output state with summation gallop Hepatomegaly, spleenomegaly seen in acute phase, epistaxis, blood tinged sputum may be seen.
• Fever – Chill phase – rigors, rising temperature, hypermetabolism Flush phase – falling temperature, diaphoresis & decreased circulating blood volume. Patients may have bouts of fever with or without spirochetemia in the first 1 week of convalescence. One to two relapses can be seen in louse borne RF up to 10 relapses in tick borne RF.
• Differential diagnosis – • Typhus fever, typhoid fever, non typhoid salmonellosis, malaria, dengue,TB, leptospirosis, viral haemorrhagic fevers. Doxycycline or tetracycline • Treatment – Erythromycin or chloramphenicol Supportive care
Lab diagnosis • Relapsing Fever • Sensitivity of staining method is 70% – Thin & thick smear examination • Thin smear Giemsa staining • Thick smear Leishman staining Dark field microscopy
Clinical features • Lyme’s disease • • • Stage 1 – tick bite elicits classical expanding erythematous skin lesion. – Spirochete cultivation at this stage is relatively easy Stage 2 – disseminated infection. – May present with acute arthritis, purulent meningitis. – secondary skin lesions , infection of the eye, Hepatitis, Myocardial damage may occur Stage 3 (Chronic phase) – Chronic skin lesions – Chronic neurologic symptoms – Chronic arthritis – Severity of arthritis may come down with every episode
• Lyme’s disease • Examination of clinical specimens isolates from skin lesions, blood, CSF & synovial fluid • Chances of cultivation of Borrelia burgdorferi minimal using BSK medium • INDIRECT IMMUNOFLUORESCENT ANTIBODY TEST – Increase in IgM from 3-6th week of illness confirms diagnosis – Increase in IgG is slow & sustained which can be used as a measure of chronicity – Persistant rise in IgM indicates persistent infection • Rarely animal inoculation methods are used.
• Vincent’s angina • Microscopic examination of stained smears if exudates from the ulcers. • Fusiform bacilli with gram negative bacilli confirms diagnosis • Fusospirochetosis. • Culture – swabs of exudates from mouth lesion inoculated into digest broth (Hartley’s broth enriched with ascetic fluid). Culture is incubated anerobically at 28-30°C. Culture is examined daily for growth.
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