Laboratory diagnosis in infections produced by

Treponema, Borrelia, Leptospira

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)

Spirochaetes

Spirochaetes

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

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

Primary syphilis: chancre on penine mucosa

Secondary syphilis: generalised rash

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

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)

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!!

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

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

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);

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

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

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)

Genus Borrelia

• Clinical significance: vector borne diseases

• Relapsing fever (Borrelia recurrentis) – lice, ticks

• Lyme disease (Borrelia burgdorferi) - ticks

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

Blood smears in relapsing fever - Borrelia

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

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)

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)

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

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)

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

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

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

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)

Leptospira – dark field microscopy

Leptospira - immunofluorescence

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