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.

1. Laboratory diagnosis in infections
produced by
Treponema, Borrelia, Leptospira

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)

3. Spirochaetes

4. Spirochaetes

5. GenusTreponema
• Comensals: human upper respiratory mucosa, genital
mucosa (e.g. Treponema phagedenis) – cultivable
• Pathogens: non-cultivable
• Treponema pallidum
– ssp. pallidum – syphilis - STI
– ssp. endemicum – bejel (endemic syphilis) – Africa, Australia
– ssp. pertenue – pian – granulomatous skin lesions →
degenerative lesions in lymph nodes and bones
• Treponema carateum – pinta – persistens skin lesions
(papulae) → mutilating scars

6. Treponema pallidum
• Causative agent of syphilis
• Transmission via:
– sexual intercourse
– mother to child (transplacentar, intra-partum) – congenital
syphilis
• Evolution stages (if untreated):
– Primary: 15 days after infecting contact; chancre – painless
ulceration at the entry site (penis, vagina, oral mucosa)
– Secondary: 45 days after chancre; skin rash
– Tertiary: after latency of 5-15 years; destructive lesions of CNS,
cardiovascular, muscles, bones, etc

7. Primary syphilis: chancre on penine mucosa

8. Secondary syphilis: generalised rash

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)

18. Genus Borrelia
• Clinical significance: vector
borne diseases
• Relapsing fever (Borrelia
recurrentis) – lice, ticks
• Lyme disease (Borrelia
burgdorferi) - ticks

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

20. Blood smears in relapsing fever - Borrelia

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)

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)

31. Leptospira – dark field microscopy

32. Leptospira - immunofluorescence

33. Leptospirosis
- Bacteriologic diagnosis - continued
• Culture media: liquid/semisolid media containing blood +
bovine serum + other ingredients
– 1st week – blood culture (rarely positive growth)
– After 1st week – urine culture, experimental disease in animals
– Incubation at room temperature, dark, very slow growth (6-14
days, up to 3-4 months) – not adequate for diagnostic purposes
• Serology:
– microscopic agglutination with Leptospira antigens
– ELISA