Institute of Medical Microbiology Semmelweis...
Transcript of Institute of Medical Microbiology Semmelweis...
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06 December 2013 1
Spirochaetales
PROF. KÁROLY NAGY
Institute of Medical MicrobiologySemmelweis University
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Taxonomy� Spirochaetales ordo
� Spirochaetae classis
�Spirochaetaceae familia�Treponema genus �Borrelia genus
�Leptospiraceae familia� Leptospira genus
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Leptospira spp. Borrelia spp. Treponema pallidum
Size [mikron] 0,1×6–20 0,2–0,5×3–30 0,2×5–15
many smooth-waved
winding with two terminal
hooks
irregular, rough windings compact, regular windings
Stainingimmune fluorescent
staining only
Native prep
Cellwall Gram-negative ? ?
Membranes
Capsule
Flagellae
Motility
Morphological properties
endoflagellae anchored at the pole
vigorous and characteristic: rotation, flection, locomotion
Shape
stained readily with aniline dyes and silver
impregnation
well visible with dark-field microscopy
spiral formed (corkscrew shaped), flexible
outer and inner (cytoplasmic) membranes
glucoseaminoglycane
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Morphology (ELMI)
Leptospira genus
Treponema genus
Borrelia genus
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Biological properties in common
� Reproduction: binary fission
� Generation time: relatively long: 17-20 h
� High sensitivity for environmental factors
⇒ transmission (!)� Narrow temperature optimum
� Narrow pH optimum
� Poor osmotic resistance
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Leptospira spp. Borrelia spp. Treponema pallidum
Organic culture media Cultivable Cultivable Non-cultivable
Nutrient requirements fastidious fastidious ?
Time 8–13 weeks 4 days–6 weeks ?
Atmospheric
requirementsAerobic Microaerophilic (Anaerobic)
Temperature optimum
[°C]28–30 30–33 (30)
Cell or tissue culture Cultivable Cultivable Non-cultivable
Animal inoculation Many lab animals Many lab animals Only in rabbit testis
Culture of spirochaetae
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06 December 2013 7
Treponema pallidum
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Historical background: Syphilis
� Colombus, America 1492
� Transfer of syphilis to Europe in October 1492
� Spreading across the entire continent within short time
� Famous infected people: Nietzsche, Schubert, Heine, Gauguin, Hoffman, Maupassant
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Historical background:the pathogen
� Discovery of T. pallidum, 1905� Fritz Richard Schaudin� Erich Hoffmann
� First diagnostic test, 1906� August Paul von Wassermann� Wassermann-reaction (complement
fixation)
� First effective treatment, 1907� Paul Ehrlich: salvarsan (arsephenamine)
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Antigenic properties
� Surface antigens: mucopolysaccharides� Treponema specific hyaluronic acid layer� Host specific hyaluronic acid & chondroitine
sulphate layer� At distal end mucopolysaccharidase enzyme
activity
� No extracellular products
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Virulence factors
� Mucopolysaccharides: adhesion
� Mucopolysaccharidase: adhesion, invasion
� Motility: invasion
� Intracellular survival in epithel, endothel cells, fibro-, granulocytes & macrophages
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Immunity
� Treponema specific antibodies� IgM: Week 1–several weeks
� IgG: Week 2–many years (also after healing)
� No long term protective effects
� Lipid specific, cross reacting antibodies� Characteristic but not specific (!)
� Directed against mitochondrial lipid antigens
� Formed on tissue decomposition: eg. syphilis, collagen diseases, tumours, malaria, tbc, pregnancy
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Natural history of immunity
� Sy I� Antibody, T-cell response
� Certain level of protection: rare re-infection
� Sy II� High titre of antibodies, intensive T-cell response
� Effective protection against re-infection
� No protection against bacterial invasion
� Latency� Mainly T-cell response
� Late latency: patient is non-infective
� Sy III� T-cell mediated immune pathological processes
� Gumma, perivascular lymphocytic infiltration
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Clinical characteristic: Syphilis I
� Primary lesion: hard ulcer (chancre) at portal of entry
� Lymphadenopathy
� Lymphangiitis syphilitica (penis: chorda syphilitica)
� Oedema induratum (bell-hammer penis)
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Penile chancrehard ulcers in the shaft of the penis
OBNI V.V.
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Vulval chancre
OBNI V.V.
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06 December 2013 17
Anal chancre
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06 December 2013 18
Chancre on the face
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Clinical characteristics: Syphilis II
� Bacteriaemia, generalised lymphadenopathy, occasionally fever
� Diverse skin affections
� Roseola (macula)
� Papula, condyloma lata
� Mucous plaques
� Syphilitic leucoderma
� Syphilitic alopecia
� Malignant syphilis (HIV!): ulcerating nodes, fever, meningeal signs
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Maculopapular skin lesions
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06 December 2013 22
Papulo-squamous lesions
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06 December 2013 23
Papulo-pustular lesions
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06 December 2013 24
Condyloma lata
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06 December 2013 25
Syphilitic alopecia
OBNI V.V
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Clinical characteristic: latency
� Positive serological reactions
� Early latency
� ≤ 1 year
� Patient is infectious
� Late latency
� > 1 year
� Patient is non-infectious
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Clinical characteristics: Syphilis III
� Granulomatous tissue reactions in different organs
� Skin: gumma
� Cardiovascular sy: syphilitic aortitis, aneurysma
� Neurosyphilis� Meningitis
� Menigovascular syphilis
� Progressive paralysis (psychiatric symptoms)
� Tabes dorsalis (spinal cord lesions)
� Gumma (space occupying process)
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06 December 2013 28
Ulcerative gumma
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06 December 2013 29
Aortic valve lesion
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Congenital syphilis at birth
� No signs of adult syphilis stage I
� Systemic involvement
� Intrauterin atrophy, death
� CNS involvement
� Anaemia
� Non-immune hydrops
� Pneumonia alba
� Hepatosplenomegaly, jaundice
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Hepatosplenomegaly at birth
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06 December 2013 32
Osteochondritis
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06 December 2013 33
Vesicular lesions, runny nose
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Late congenital syphilis
� ≥ 2 years of age� Hutchinson-triad
� Keratitis parenchymatosa, blindness� Central deafness� Hutchinson-teeth (peg-shaped upper incisors)
� Short maxilla� Saddle nose� Gumma� Bone deformities� Mental retardation, convulsions
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06 December 2013 35
Hutchinson-teeth
Peg-shaped upper incisors
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Short maxilla, saddle nose
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06 December 2013 37
Ulcerative gumma: perforation of the palate
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Microbiological diagnosis of syphilis
� Direct microscopic examination
� Sample: chancre discharge, wet lesions
� Method: dark-field microscopy
� Serology
� Non-specific (reagin) type antibodies
� Specific (anti-treponemal) antibodies
� (Culture: not possible)
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06 December 2013 39
Dark-field microscopy 1,000×
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Syphilis serology
� Non-specific (reagin) antibodies� Antigen: cardiolipin, Reiter-protein
� Wassermann, VDRL (control of therapy),
� RPR (rapid plasma reagin)
� Specific (anti-treponemal) antibodies� Antigen: T. pallidum
� TPHA: screening test (IgM+IgG)
� FTA-Abs: verification (IgM, IgG)
� TIT (Nelson)
� Most recently: ELISA, immune blot (IgM, IgG),LIA
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06 December 2013 42
VDRL/RPR (rapide plasma reagin)
TPHATreponema PallidumHaemagglutination test
agglutination
precipitate
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06 December 2013 43
Detection of T.Pallidum antibodies by
Western-blot
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Treatment of syphilis
� Drug of choice: penicillin G
� High dose
� Long course of treatment
� Alternative drugs
� Ceftriaxon, erythromycin, doxycyclin
� Jarisch-Herxheimer reaction
� Toxic material from dead bacteria
� Interleukines, cytokines
� Fever (40 °C), enhanced exanthema, shock
� Treatment: glucocorticoides
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Prevention of syphilis
� Individual level
� Safe sex, avoiding promiscuity
� Avoiding occupational exposition: plastic (latex) gloves
� Population level
� Effective education
� Mass screening
� Exploration & treatment of infected individuals
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06 December 2013 46
Streamlined posters: Swiss, 1990’s
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Borrelia genusBorrelia genus
Species Disease association
B. burgdorferi sensu lato Lyme-borreliosis
B. burgdorferi sensu strictoErythema chronicum migrans,
Lyme-arthiritis
B. gariniiPolymeningoradiculitis
(Bannwarth's disease)
B. afzeliiAcrodermatitis chronica
atrophicans
B. recurrentis
B. duttoniiFebris recurrentis (Relapsing fever)
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Borrelia burgdorferi sensu lato
B. burgdorferi 400× ELMI
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Occurrence
� Natural reservoir
� Red deer
� Wild rodents
� Arthropod vector & reservoir: ticks (Ixodes)
� I. scapularis (Eu), I. pacificus (USA), I. persulcatus (Asia)
� Prevalence: 30–50%
� Vertical (transovarial) transmission
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Pathogenesis
� Portal of entry, local reproduction of bacteria
� Spreading with blood stream
� Organ manifestation (skin, joints, heart, liver, spleen, CNS)
� Perivascular infiltrate with lymphocytes / plasma cells (like in immune complex vasculitis)
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Clinical characteristics:Lyme-borreliosis
� Incubation period: 2 days–4 months–? years� Skin
� Erythema chronicum migrans (ECM)� Lymphadenosis benigna cutis (LBC)� Acrodermatitis chronica atrophicans (ACA)
� Arthritis� Carditis� Neuroborrelosis
� Peripheral neuritis (bilateral n. VII, rarely n. VI palsy)� Serous meningitis� Bannwarth-syndrome
� Prognosis: excellent if treatment started early
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Lyme-borreliosis: ECM
Erythema chronicum migrans
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Lyme-borreliosis: ECMErythema cronicum migrans
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Microbiological diagnosis
� Serology
� Sample: serum and/or CSF
� Routinely: ELISA (IgM, IgG)
� Verification: Western-blot
� Detection of bacterial DNA
� Sample: biopsy material, CSF, synovial fluid
� Method: PCR with hybridisation (!)
� (Culture: for research purposes only)
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Treatment
� ECM, LBC, ACA, arthritis
� Penicillin-V orally for 10 days
� Doxycyclin orally for 30 days
� Carditis
� Penicillin G high dose iv. for weeks
� Neuroborreliosis, arthritis
� Ceftriaxon iv. (3. generation cephalosporin) for weeks
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Prevention
� Specific: vaccination approved byUSA FDA (Osp A): Lymerix®
� Non-specific
� Prevention of tick bite
� Use of repellents
� Correct clothing
� Correct and early removal of ticks
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Correctly selected clothing
Removal of ticks
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B. recurrentis, B. duttonii
� Reservoir: humans, wild rodents (asymptomatic infection)
� Arthropod vector� B. recurrentis: human body loose (human-human)� B. duttonii: soft ticks, Ornithodoros spp. (animal-
human)
� Pathogenesis: antigen shifts� Disease association: Febris recurrentis
(Relapsing fever)� B. recurrentis: epidemic (war, poverty)� B. duttonii: endemic
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Consequence of B. recurrentis antigen shifts
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Microbiological diagnosis
� Dark field microscopy
� Blood film
� Culture
� Animal inoculation
� Identification with fluorescent antibodies
B. recurrentis in blood film 400×
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Treatment & prevention
� Antibiotics: doxycyclin, erythromycin
� B. recurrentis: usually single dose cures
� B. duttonii: 10-day course
� Prevention
� Specific: no vaccination available
� Non-specific: personal hygiene, control of vectors
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Leptospira genus
� Old (phenotypical) classification
� Human and animal pathogenic: L. interrogans� Several sero-groups (23) & sero-variants (200)
� Saprophytic, non-pathogenic: L. biflexa� Several sero-variants (63)
� Novel (genotypical) classification
� Leptospira genus
� Leptonema genus
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Leptospira interrogans sensu lato
Leptospira spp. scanning ELMI
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Occurrence
� Worldwide
� Natural reservoir: rodents, cattle, domestic swine, dogs
� Asymptomatic carriage in renal tubuli
� Natural water sources contaminated with animal urine
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Leptospirosis: Are you at risk?
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Transmission
� Zoonotic infection, human:
� accidental host
� Direct� Contact with urine of animals
� Indirect� Contaminated natural water sources
� Contaminated fomites
� Occupational risk (farmers, miners), laboratory accidents
� Portal of entry: skin (intact?), mucous membranes, conjunctiva
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Pathogenesis
� Establishment: portal of entry
� Invasion
� Regional lymph nodes, reproduction
� Blood stream
� Organ manifestation, intersticial space
� Tissue damage
� Week 1: no inflammatory reactions
� Week 2: inflammation (specific antibodies appear)
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Clinical characteristics
� Systemic infection with two phases� Incubation: days 7–13� Phase 1: fever & muscle pain � Phase 2: organ damage
� Meningitis, uveitis, exanthema� Pneumonitis� Renal tubular damage , immune complex
glomerulonephritis� Liver damage� Liver & kidney involvement & jaundice & pulmonary
haemorrhage & rapid progression: Weil’s disease� Lethality: 1–10–40%
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Renal damage in leptospirosis, autopsy Icteric sclera (jaundice)
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Microbiological diagnosis
� Serology
� At week 1, after symptoms develop
� Microscopic agglutination test: titre≥100 or paired sera 4×rise of titre
� ELISA (IgM, IgG)
� Culture
� Secondary significance only
� Week 1: blood, CSF
� Week 2: fresh urine
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Treatment
� Specific
� Antibiotics: penicillin G, ampicillin & doxycyclin
� Effective if given in early stage (!)
� Supportive
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Prevention
� Individual level� Avoiding contact with animal urine, frequent
hand washing
� Avoiding contaminated water
� Vaccination: formaldehyde inactivated
� Chemo-prophylaxis: doxycyclin
� Population level� Vaccination of domestic animals (eg. dogs)
� Control of rodents
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Pathogenesis
� Adhesion (mucopolysaccharides & -ase)� Local reproduction of bacteria� Invasion (mucopolysaccharidase &
motility)� Escape from immune system (intracellular
survival)� Tissue damage
� Endarteritis obliterans, periarteritis → ischemic damage
� Granuloma formation → space occupying process
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ssp. pallidum ssp. pertenue T. carateum ssp. endemicum
venereal syphilisyaws, frambözia,
pianpinta, carate
endemic syphilis,
bejel
hot, dry climate
tropical areasMiddle & South
Americadeserts
Affected age adultschildren,
adolescentschildren children & adults
Transmission STD mucous membran
Congenital
formexists rarely
skin
none
Epidemiology of the four treponematoses
hot, humid climate
worldwidePrevalence
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First detailed description
� Girolamo Fracastoro(Hieronymus), 1530
� Educational poem
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RIT – rabbit inoculation test
Inoculation of T.Pallidum into rabbit testicle
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1 2
KE DL R IHIV POS 1997 (���� 2000.02)
HIV POS 2000.04 SY POS 1996
TpN471715
TmpA
3 4 5
Syphilis LIAHIV-1 pol LIPA
2000.11
Simultaneous exchange of Sy and HIV infection
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Posters: USA, 2nd Word War
Hitler VD Hirohito
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Posters, USA, 1950’s
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Posters: Great Britain, 1950’s
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Posters: Great-Britain, 1950’s
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Streamlined posters: USA, 1970’s
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Historical background� Obermeier (1873): B. recurrentis
� Burgdorfer (1982): B. burgdorferi
� Name of the genus: Borell (1867–1936)
� Lyme-borreliosis: Lyme, Old-Lyme
Connecticut, USA (1974, 1975)
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Immunity
� Specific T-cell & antibody response
� No reliable, long term protection after infection
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Virulence factors
� Hyaluronidase
� Motility
� Haemolysins (L. interrogans serovar. grippotyphosa, pomona)
� Exotoxins (?)
� Endotoxins (?)
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Immunity
� Specific T-cell & antibody response → significance is little known� Specific antibodies appear at the end of
week 1 (acute serum)
� Highest titre at weeks 2–3 (reconvalescent serum)
� Immune pathological processesat phase 2