This document discusses the laboratory diagnosis of infections caused by Treponema, Borrelia, and Leptospira bacteria. It describes the characteristics and pathogenic species of each genus, how they cause diseases like syphilis, Lyme disease, and leptospirosis, and the methods used to diagnose infections through direct examination of clinical samples, culture techniques, and serological tests. Key diagnostic tests include darkfield microscopy, immunofluorescence, culture media, microscopic agglutination, ELISA and Western blot. Penicillin and doxycycline are commonly used for treatment.
2. Order Spirochaetales
• Family
Spirochaetaceae
– Genera:
• Treponema
• Borrelia
• Family
Leptospiraceae
– Genus Leptospira
• General characters:
– Long (up to 250 µm),
thin (0.1-0.6 µm
diameter),
spirral/helically coiled
– High motility
(flagella/axial
filaments)
9. Tertiary syphilis (patient is not infectious)
• 3 forms:
– gummatous (15%): gummas = soft, tumor-like balls of
inflammation on the skin, bones, and liver
– neurosyphilis (6.5%):
• Early: meningitis
• Late: general paresis / tabes dorsalis
(myelopathy), dementia
– cardiovascular (10%): aortic aneurisms
10. Treponema pallidum
- Laboratory diagnosis -
Collection of specimens:
• chancre secretion (primary syphilis)
• secretion from skin lesions (secondary syphilis)
– choose most recent lesion, remove crust, press lesion in order to
cause bleeding, collect serous exudate
• blood for serlogy (all stages)
Specimens from lesions must be examined asap
(treponemae are not viable for a long time outside the
body)
11. Treponema pallidum
- Microscopy -
• Wet mount in dark
field/contrast phase:
• shining treponemae, highly
motile on the dark background
of microscopic field
• Stained smears: e.g. Silver
staining
ATTENTION: oral specimens
might contain comensal
treponemae!!
12. Treponema pallidum
- Serology -
Diagnosis based on antigenic structure:
– Cardiolipinic Ag – present in all treponemae + other bacteria
– Proteic Ag (Reiter) – Genus-specific (present in all treponeame)
– Treponema pallidum specfic Ag
Diagnostic tests:
• Nonspecific (nontreponemic):
– VDRL (flocculation)
– Bordet-Wasserman reaction (complement fixation)
• Specific (treponemic):
– TPI (Treponema pallidum immobilization) test
– passive hemagglutination
13. Treponema pallidum
- Serology - continued
Nonspecific, nontreponemic tests:
VDRL
(Veneral Disease Research
Laboratory, Atlanta, USA)
Principle: antibodies (anti-
cardiolipin Ab) produced by a
patient with syphilis react with
an extract of ox heart; reaction
visualized through foaming of
the test tube fluid, or
"flocculation".
Patient Ab react with bacterial
components
14. Treponema pallidum
- Serology - continued
Nonspecific, nontreponemic tests (continued):
Bordet-Wasserman test:
Principle: Ab in patient serum will inactivate serum
complement in the presence of ”reagines” (produced by
infected tissues in response to bacterial infection);
15. Treponema pallidum
- Serology - continued
Specific treponemic tests
TPI (Treponema pallidum immobilization):
• Principle: specific anti-Treponema pallidum Ab in patient
serum, in the presence of complement, immobilize
actively motile T. pallidum obtained from testes of syphils
infected rabbits
Passive hemagglutination:
• Principle: specific anti-Treponema pallidum Ab in patient
serum cause agglutination of treponemic Ag adsorbed
on the surface of red blood cells
16. Treponema pallidum
- Antibiotic sensitivity -
• Penicillin – i.v.
• Alternatively (in case of allergy):
– Doxycycline, tetracycline, azithromycin
– OR – desensitization – to enable
administration of penicillin
• Pregnant women with syphilis must
receive penicillin to prevent congenital
syphilis
17. Order Spirochaetales
• Family
Spirochaetaceae
– Genera:
• Treponema
• Borrelia
• Family
Leptospiraceae
– Genus Leptospira
• General characters:
– Long (up to 250 µm),
thin (0.1-0.6 µm
diameter),
spirral/helically coiled
– High motility
(flagella/axial
filaments)
19. Borrelia recurrentis
• Main causative agent of relapsing fever (also caused by
other agents e.g. Rickettsia)
• Vectors: human lice (Pediculus corporis) + certain
species of ticks
• Clinical aspect: sudden fever, chills, headache, nausea -
for 2-9 days; symptoms reappear after 3-10 days;
evolution continues with similar cycles
• Laboratory diagnosis:
– detection of spirochetes in blood smear
– ELISA – detection of specific Ab in patient serum
21. Borrelia burgdorferi
• Clinical significance: Lyme disease (boreliosis) – disease
described for the 1st time in Lyme Connecticut USA
1976)
• Reservoir: birds, dogs, horses
• Transmission via tick bites; incubation 1-3 weeks
22. Lyme borreliosis – ”disease with 1000 faces”
- stages -
I. Erythema migrans:
• 3-30 days after tick bite;
• ”bull‘s eye” rash; may further
appear on other parts of the body
II. General dissemination:
myocarditis, arthritis,
lymphadenitis, neurologic
symptoms (meningitis,
meningoradiculitis)
III. Chronic relapsing arthritis (knee,
elbow), chronic atrophic
acrodermatitis (skin sclerosis and
atrophy of limbs → generalised)
23. Borrelia burgdorferi
- Laboratory diagnosis -
• ELISA for the detection of specific Ab (IgM and IgG) in
patient serum
• Confirmatory tests: immunoblot (Western blot)
• Specific choice and sequence of tests – depends on
stage of disease (described in diagnostic guidelines)
24.
25. Lyme disease
TREATMENT:
• early stages: doxycycline, amoxicillin, cefuroxime
• later stages: intravenous ceftriaxone or penicillin
PREVENTION:
• Prevention of tick bites
• Safe removal of ticks
• http://www.cdc.gov/lyme/prev/index.html
26. Order Spirochaetales
• Family
Spirochaetaceae
– Genera:
• Treponema
• Borrelia
• Family
Leptospiraceae
– Genus Leptospira
• General characters:
– Long (up to 250 µm),
thin (0.1-0.6 µm
diameter),
spirral/helically coiled
– High motility
(flagella/axial
filaments)
27. Genus Leptospira
General characters:
• aerobic, helicoidal, flexible, 6-20 µm length, 0.1 µm
diameter, motile, terminal “hook” at each end
Species:
• Leptospira interrogans – pathogenic
– Serotypes: L. canicola, L. icterohemorrhagiae, L. pomona, etc
• Leptospira biflexa – saprophitic – present in water
28. Genus Leptospira
- Clinical significance -
• Zoonosis – domestic and wild animals
• Human infection:
– contact with water, soil - contaminated with animal urine
– occupational exposure: veterinarians, farmers, field workers,
hunters, etc
– Germs may penetrate intact skin / microlesions → rapid blood
dissemination → organs (liver, kidney, eye), CSF
• Disease – Leptospirosis: mild clinical forms → severe
icteric disease with hepatic & renal disfunctions
29. Leptospirosis
- continued -
• Incubation: 3-30 days
• First stage: germs present in blood and CSF ~ 1 week
• Further stages: germs eliminated in urine – from 2nd
week; urine elimination persists 2-3 months
• Laboratory diagnosis:
– Bacteriology
– Serology
– Experimental disease
30. Leptospirosis
- Bacteriologic diagnosis -
• Collection of specimens: blood, CSF, urine, peritoneal
fluid
• Specimens must be centrifuged to increase the chance
of direct detection in sediment
• Microscopy:
– Wet mounts examined in dark field
– Immunofluorescence (fluorescent antibodies)