SPIROCHAETALES. Katalin Kristóf 2014

Transcription

1 SPIROCHAETALES Katalin Kristóf 2014

2 Spirochaetales 2 Familiae Spirochetaceae Treponema Borrelia Leptospiraceae Leptospira Common feature: thin, coiled, spiral shaped bacteria Length: µm Too thin (0,1-0,2 µm) to be seen with light microscopy stained with Gram => Darkfield illumination; IF; silver-impregnation periplasmic flagellae (endoflagellae)

3 Spirochaetales Associated Human Diseases Genus Species Disease Treponema Borrelia pallidum ssp. pallidum pallidum ssp. endemicum pallidum ssp. pertenue carateum burgdorferi recurrentis Many species Syphilis Bejel Yaws Pinta Lyme disease (borreliosis) Epidemic relapsing fever Endemic relapsing fever Leptospira interrogans Leptospirosis (Weil s Disease)

4 Treponemal infections Apathogen: T. minutum, T. reiteri, T. denticola, T. phagedenis Plaut-Vincent angina

5 Nonvenereal Treponemal diseases (treponematosis): Treponema pallidum ssp. endemicum bejel (endemic syphilis) - spread person to person by contaminated eating utensils - initial oral lesions, secondary skin lesions - Africa, Asia, Australia (endemic) Treponema pallidum ssp. pertenue yaws - granulomatosous disease, skin lesion - South America, Central Africa, Southeast Asia Treponema carateum pinta - skin lesions spread by direct contact with infected lesions - South America Lues tests positive!

6 Vincent s angina Ulcerative tonsillitis causing tissue necrosis often due to extension of acute ulcerative gingivitis Fusobacterium nucleatum in combination with oral spirochates (Treponema vincentii and others) causes the fusospirochaetal infections

7 Treponema pallidum Morphology: - thin, coiled spirochetes (0,1 to 0,2 X 6-20 µm) - Three periplasmic flagellae are inserted at each ends (endoflagellae) - Outer membrane proteins: - TrompI, TrompII, TrompIII - Inner proteins:15kda, 17kDa, 45.5kDa, 47kDa (endoflagellum, cytoplasma membrane, cytoplasma) Multiplication: by binary transverse fission Cultivation: can not be cultivated on cell-free artificial culture media - Kept alive in rabbit testis! - rabbit epithelial cells (GT 30h, only a few generations)

8 Treponema pallidum virulance factors, pathogenesis Outer membrane proteins promote adherence to host cells Hyaluronidase may facilitate perivascular infiltration Coating of fibronectin protects against phagocytosis Tissue destruction primarily results from host s immune response to infection Destroy cytoplasma membrane, mitochondrial membrane => Cholesterol, Lecithine, Kardiolipin Ag free ( RPR, VDRL) Endarteriitis, Periarteriitis» inflammation, necrosis T-cell dependent late hypersensitivity»granuloma Gumma

9 I. Acquired syphilis (venereal disease) Spread: sexually (STD) The great imitator! 3 phases: 1) primary phase 1-2 weeks incubaton period - The initial syphylitic chancre develops at the site where the spirochete is inoculated - Generally on the genitalia, rare: oral cavity, perianal region - localised replication of bacteria =>Papule, macule =>erodes => chancre ulcus durum: hard, painless ulceration - Painless lymphotic nodes bubo indolens - mucocutan lesion is very infectious! - spontaneous remission may occur after 2-6 weeks (50%)

10 2. Stage develops after 4 to 8 weeks from the primer infection (haematogen spreading) disseminated disease - with generalised mucocutaneous rash, - superficial sores (mucous patches) may occur on mucous membranes of the mouth, vagina, or anus, - while wart-like lesions called condylomata lata may form in moist intertriginous areas. - Hepatitis - Neurological signs - High fever - micropolyadenopathy - spontaneous remission may occur; after 1-2 years these symptoms can reoccur latent persistence Highly contagious!

11 3. Stage (late phase) after 3-30 years from the primer infection - all tissues are involved - gumma (granulomatous lesions) in bone, vessels, skin - neurosyphilis: tabes dorsalis, paralysis progressiva (encephalopathy), ataxia, dementia, N. opticus degeneration - cardiovascular syphilis: aortitis, aorta aneurysm (rupture)

12 Progression of Untreated Syphilis Late benign Gummas in skin and soft tissues Tertiary Stage

13 II. Congenital (connatal) syphilis T pallidum subsp pallidum also damages foetuses (from 4 gw) Approximately 50 percent of foetuses are aborted or stillborn; In early congenital syphilis (before the age of two years ~ II): mucocutaneous lesions, osteochondritis, anaemia, and hepatosplenomegaly. In late congenital syphilis (> 4 years, ~III): interstitial keratitis and blindness, tooth deformation (notched incisors and moon molars), eighth-nerve deafness, neurosyphilis, rhagades (fissures at mucocutaneous junctions), cardiovascular lesions, Clutton's joints (fluid accumulation on knee), and bone deformation of the legs, nasal septum, and hard palate. Can be preventing with penicillin treatment of the Treponema infected pregnant woman!

14 early congenital syphilis

15 late congenital syphilis Hutchinson's triad: interstitial keratitis, notched incisors and eighth-nerve deafness Plexus brachialis paralysis Tibia deformation Hutchinson s teeth the incisors are smaller than normal, with sloping sides and central semilunar notches.

16 Diagnosis Sample: exudates, punctuates from the mucocutan lesion, (1-2 phase) Microscopy: Too slender (0,1-0,2 µm) to be seen with light microscopy stained with Gram Live treponemes can be visualized by using dark-field microscopy (rapid rotation about its longitudinal axis and bending, flexing, and snapping about its full length) Fluorescent labelled antitreponemal antibodies Silver-impregnation PCR Serology

17 Serology I. Non specific treponemal tests reaginic antibodies developed against lipids released from damaged cells during the early stage of disease and present on the cell surface of the treponema KKR VDRL RPR Ag: Cardiolipin (from extraction of beef heart), lecitin, cholesterol II. Specific treponemal tests detection of specific - Immobilizin Ab (treponemal) TPIT FTA-Abs TPHA, TPPA TP-ELISA Western Blot, Immunoblot Treponemal Ag: TP proteins, lipoproteins

18 I. Non treponemal tests VDRL-test (Venereal Disease Research Laboratory) - Ag = freshly prepared cardiolipin suspension - patient's serum is inactivated at 56 oc, for 30 min - a drop of the cardiolipin suspension is placed on a glass slide - mixed with a drop of the inactivated serum Negative: Cardiolipin suspension remain dispersed. Positive: Cardiolipin forms visible clumps when combining with reagin. RPR test (Rapid Plasma Reagin) Ag = cardiolipin suspension attached to latex particles patient's serum should not be inactivated Negative: Cardiolipin-latex suspension remains intact. Positive: Cardiolipin-latex is agglutinated and sediments as rough granula - Flocculation

19 Flocculation: granules => Ab equally Ag Dilution of patient serum!

20 II. (Specific) treponemal tests 1. FTA-ABS = Fluorescent Treponemal Antibody-absorption Ag = killed, fixed T.pallidum on glass slide Overlayed with the patient s serum, which has been mixed with an extract of nonpathogenic treponemes (T. reiteri) fluorescein labelled antihuman immunglobulin (IgG, IgM) fluorescein microscopy The most sensitive and specific /2.TPI = Treponema pallidum immobilisation test living T.pallidum = Antigen inactivated patient s serum, complement of guinea pigs The reaction is based on that T. pallidum cells are inhibited in their movement if they are exposed to specific IgG antibodies in the presence of complement. Negative: T. pallidum cells exhibit locomotion. Positive: T. pallidum cells do not show movement. /

21 II. Treponemal tests 3.TPHA, TPPA T. pallidum haemagglutination test Bird red blood cells sensitized with T. pallidum antigens (E.coli Tp15, Tp17, Tp 47 - recombinant) Neg/Pos (1:80) Titer IgG, IgM and IgA T. pallidum particle agglutination Gelatin particles sensitized with T. pallidum antigens (Bacillus subtilis Tp15, Tp17,Tp47 - recombinant) Neg/Pos (1:80) Titer IgG, IgM and IgA

22 II. Treponemal tests: 4. TP-ELISA Rekombinant Ag-s Tp15, Tp17, Tp47

23 II. Treponemal tests: 5. Western blot Immunoblot strip 15, 17, 45.5 and 47 kda recombinant proteins IgG, IgM

24 BAP (biologically aspecific positivity) :

25 Sensitivity of serological tests in untreated syphilis Test Primary Secondary Latent Tertiary VDRL 78 (74-87) (88-100) 71 (37-94) RPR 86 (77-99) (95-100) 73 FTA-ABS 84 (70-100) Treponemal Agglutination 76 (69-90) (97-100) 94 EIA The use of only one type of serologic test is insufficient for diagnosis.

26 Summary of serological tests: For screening TPPA/TPHA FTA-Abs Tp-ELISA For verification Western blot RPR/ VDRL Determination of the stage To monitor the effectiveness of therapy To detect reinfection

27 Treatment, control penicillin treatment eradicates all stages, including congenital infection in pregnancy /Doxycyclin, Azithromycin/ Jarisch-Herxheimer lysis of the treponemes causes the release of huge amount antigenes => high fever, anaphylaxia, abortion (steroid) Prevention: safe sex For sex partners of patients with syphilis in any stage: Draw syphilis serology Perform physical exam Congenital - Can be preventing with penicillin treatment of the Treponema infected pregnant woman screening!

28 Epidemiology of Borrelia Infections Borrelia recurrentis Pediculus humanus Borrelia spp. Ornithodoros spp. Borrelia burgdorferi Ixodes spp.

29 Borrelia genus Morphology: 4-18 µm, spirochete, have fewer coils. Seven to twenty periplasmic flagella originate at each end and overlap at the center of the cell - twisting motility.

30 Borrelia recurrentis Reservoir: humans Vector: human body louse Disease: 1.Epidemic (louse-borne) relapsing fever = periodic febrile and afebrile cycles bacteria undergo antigenic variation specific IgM complement-mediated lysis bacteria disappear from the blood hidden borrelia can change their outer proteins by gene rearrangement emerge as novel organism risk: exposed to lice crowded, unsanitary conditions (war, natural disasters)

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32 2. Endemic (tick-borne) relapsing fever: many Borrelia species (Borrelia hermsii) Reservoir: rodents (zoonotic infection) Vector: soft-shelled tick Symptoms: fever = bacteremia 1 week afebrile period fever returns Diagnosis: Sample: blood (during fever) Giemsa staining Treatment: tetracycline, erythromycin

33 Diagnosis: Sample: blood (during fever) Giemsa staining Antibody detection by indirect immunofluorescence assay Cultivation: slow growth on artificial culture media microaerophilic, complex nutrition requirement Cultivation does not used to be successful Treatment: tetracycline, erythromycin Control - by avoiding the vectors (should wear clothing that covers as much of the skin as possible and use tick repellents)

34 Borrelia burgdorferi Reservoir: deers, rodents Vector: hard-shelled tick (Ixodes ricinus Europe) Lyme disease: 1977 group of children develop arthritis in Lyme (Connecticut) 1982 W. Burgdorfer discovered spirochete responsible for the disease

35 Clinical symptoms 1.Localised: erythema chronicum migrans /ECM/ - skin lesion due to tick bite - flat, red border macule with central clearing as it develops - ranging from 5 cm to 50 cm - fades and disappeares within weeks 2. Early disseminated stage - within days and weeks, untreated patient - hematogenous dissemination - headache, fever, arthalgia, myositis - heart failure (heart block, myopericarditis) - meningitis, encephalitis 3. Late manifestation - within months and years - acrodermatitis chronica atrophicans (ACA) /in elderly/ - arthritis - lymphadenomatosis benigna cutis /in children

36 Lyme Borreliosis Erythema chronicum migrans Acrodermatitis chronica atrophicans Lymphadenosis benigna cutis Lyme arthritis

37 Diagnosis - clinical symptoms (ECM) - low number of B. burgdorferi in mucocutan lesions - from mucocutan lesion nucleic acid amplification (PCR) SEROLOGY is important! Immunofluorescence assay ELISA Western blot confirmation of ELISA Treatment: Early Lyme disease: tetracyclines, penicillins. Arthritic and neurologic disorders: high-dose intravenous penicillin G or ceftriaxone. Prevention: avoid ticks protective clothing, insect repellents The spirochetes are present in the midgut of the tick, and 12 to 24 hours is required before the spirochetes are transmitted.

38 Morphology - thin, coiled spirochete, 20 µm, both ends hook shaped Leptospira genus

39 Pathogenic species: Leptospira interrogans many serovariants (serologically distinct groups) serovariants different animal reservoirs L. interrogans pomona swine L. interrogans grippotyphosa mouse L. interrogans icterohaemorrhagiae rat L. interrogans canicola dog Asymptomatic infection in reservoir hosts colonise the renal tubules - spread by the urine of the infected animal streams, standing waters can be contaminated Humans are accidental hosts: leptospira can penetrate the skin Occupational infections: farmers, veterinarians, agricultural workers

40 Disease: leptospirosis (zoonosis) Asymptomatic infection Symptomatic infection: Typical biphasic disease 1-2 weeks incubation 1) Acut :flue like symptoms (fever, muscle pain) leptospira in the blood 2) Immune leptospiruric phase: sudden onset of myalgia, headache, abdominal pain, conjunctival suffusion, vascular collapse, thrombocytopenia, hepatic, renal failure aseptic meningitis (no pus in the cerebrospinal fluid) Weil s disease: severe form of leptospirosis (mortality: 15-20%) (progressive impairment of hepatic and renal function ; death) Hepatic involvement with jaundice (icteric disease) (The first human case of leptospirosis was described in 1886)

41 Clinical Progression of Icteric (Weil s Disease) and Anicteric Leptospirosis (pigmented part of eye)

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43 Diagnosis Sample: blood, cerebrospinal fluid, urine silver impregnation Fluorescein labeled antibody cultivation - Korthof culture media,,smoke like growth Containing 10% rabbit serum (long chain fatty acids and vitamins B1 and B12; ph7,4) Slow growing (2 weeks) Low temperature (30 oc) Serology: Microscopic Agglutination Test detection of specific antibody in the patient s serum after 2 weeks - serial dilution from the patient s serum - add equal amount of living leptospira - the bacteria will agglutinate - microscope to detect agglutination

44 Treatment: doxycycline Prevention: - Doxycycline can be used as chemoprofilaxis. -Vaccination for high risk individuals (laboratory workers) is availbale. - Materials contaminated with animal urine (e.g. natural water sources) should be avoided. - Human leptospirosis can be controlled by reducing its prevalence in wild and domestic animals.

45 Köszönöm a figyelmet!