Spirochetes A short description on different spirochete including Treponema, leptospira interoggan, borrelia . With emphasis for nursing students.

1. Spirochete

2. Spirochetes
Spirochetes are thin, fexible, elongated spirally coiled gram negative bacilli.
The characteristic feature of spirochetes is that the flagella are internal. (Between the
outer membrane and peptidoglycan layer)
Endoflagella are responsible for the motility of spirochetes.
Spirochetes can swim even in highly viscous, gel-like medium.
Pathogenic spirochetes includes: Treponema, Borrelia and Leptospira.
Larger spirochetes like Borrelia are gram-negative but other spirochetes cannot be
stained by routine staining methods. However, they can be seen under dark ground
microscopy, silver impregnation method and immunofluorescence.

4. TREPONEMA
Treponemes are slender spirochetes with fine spirals having pointed ends.
Most of them are commensals in mouth and genitalia.
while few are pathogenic to men, such as:
● T. pallidum subspecies pallidum - Syphilis
● T. pallidum subspecies pertenue - Yaws disease
● T. pallidum subspecies endemicum - endemic syphilis
● T. carateum - pinta

5. TREPONEMA PALLIDUM
Treponemes are extremely thin and delicate with tapering ends.
They are flexible, spirally coiled around the long axis; possess 6-14 spirals .
They possess about 3-4 endoflagella and are actively motile exhibiting corkscrew
motility. Endoflagella are also highly antigenic.
Treponemes cannot be visualized by light microscope.

6. PATHOGENESIS OF SYPHILIS
Syphilis is one of the ancient sexually transmitted disease.
Mode of transmission:
● Sexual contact
● Blood transfusion
● Transplacental
T. pallidum rapidly penetrates through the minute abrasions on the skin or mucosa
and, within a few hours, enters the lymphatics and blood to produce systemic
infection and metastatic foci long before the appearance of a primary lesion.

7. CLINICAL MANIFESTATIONS
Syphilis
Clinically, patients suffering from syphilis pass through four stages if left untreated:
primary, secondary, latent and tertiary (or late) stages.
Primary Syphilis : It usually begins as a single painless papule that rapidly becomes
ulcerated, hard, and indurated (Single painless ulcer - hard chancre). It is very rich in
spirochetes. The most common sites are penis (in males), cervix or labia (in females),
and anal canal, rectum or mouth. The chancre generally heals within 4-6 weeks.

9. Secondary Syphilis : it usually develops 4-8 weeks after the healing of primary lesion. Skin and
mucous membranes are commonly affected and characterized by rashes. There may be
Condylomata Iata ( large mucocutaneous papules) and mucolas patches. Generalized
lymphadenopathy is also seen.
Latent syphilis is characterized by absence of clinical manifestations of syphilis with positive
serological tests for syphilis. Patients are still infectious transmitting the infection either by
bloodstream or in utero. Latent syphilis might cure spontaneously or progress to tertiary syphilis or
may persist as latent syphilis.
Tertiary Syphilis develops decades after initial infection. It may present as
● Neurosyphilis: it may present as meningitis, tabes dorsalis ( demyelination of the posterior
columns of spinal cord), or paralytic dementia (organic mental disorder)
● Cardiovascular syphilis: Characterized by aneurysm of ascending aorta and aortic
regurgitation
● Gumma : Gummas are locally destructive granulomatous lesions. They can occur in any organ
but most commonly seen in bone and skin.

11. Congenital Syphilis
A pregnant syphilitic woman can transmit Treponema pallidum to the fetus through
placenta beginning about the tenth week of gestation.
Some of the infected fetuses die and miscarriages result while others are stillborn at
term.
Some are born live but develop the signs of congenital syphilis in childhood, e.g.
interstitial keratitis, Hutchinson’s teeth, saddle nose, periostitis and many central
nervous system and structural anomalies. However, adequate treatment of mother
during pregnancy prevents congenital syphilis.

12. Laboratory Diagnosis
Laboratory diagnosis of syphilis consists of demonstration of treponemes, detection
of antibodies and PCR.
Microscopy
Specimen: clear exudate (that is not mixed with blood) from the lesion by gentle
pressure to the base.
Dark-ground Microscopy :
T.pallidum appears as slender, flexible, spirally coiled bacilli with tapering
ends.Shows corkscrew motilily. A negative results do not exclude the diagnosis of
syphilis, because of its low sensitivity.

13. Direct Fluorescent Antibody Staining for T. pallidum
Fluorescent labelled monoclonal antibody Targeted against T. pallidum surface
antigen is used here.
T. pallidum appears as distinct, sharply outlined, apple green fluorescent color
bacilli.
Silver Impregnation Staining
Treponema do not take up ordinary stains as they are extremely thin and delicate.
Treponema reduce silver nitrate to metallic silver that is deposited on the surface,
making them thicker. Then they can be stained using levaditi stain or Fontana strain.

15. Culture
Pathogenic Treponemes including T. pallidum cannot be grown in artificial culture
media.
Antibody Detection (Serology)
Depending upon the type of antigen used, two types of tests are available to detect
antibodies in patient's sera:
● Non-treponemal tests: Detect non-specific reagin antibody by using
cardiolipin antigen derived from bovine heart.
● Treponemal tests: Detect species-specific antibody by using T.pallidum specific
antigen; which is polysaccharide in nature.

16. Non-treponemal tests
1. Wassermann test : a type of complement fixation test and
2. Kahn test: a type of tube flocculation test
Both are no longer in use.
1. Venereal Disease Research laboratory (VDRL) Test
This test was named after Venereal Disease Research Laboratory (VDRL), New
York, where the test was developed .
It is the most widely used, simple and rapid serological test.

17. Procedure:
● Patient's serum is inactivated by heating at 56°C for 30 minutes.
● 0.05ml of inactivated serum is mixed with a drop of VDRL antigen and the slide
is rotated at 180 revolutions per minute for 4 minutes in a VDRL rotator and
examined under microscope.
● The results are read as follows
○ Non-reactive: Uniformly distributed fusiform crystals represent the
presence of VDRL antigen only which indicates a negative result.
○ Reactive: Presence of medium to large clumps signifies antigen antibody
complexes; hence, it indicates a positive result.
● Positive sample of VDRL should bed confirmed by specific tests such as TPHA
test. ( False positive result can occur in malaria, Typhoid, leprosy, pregnancy
etc)

19. Treponemal or Specific Tests
All reactive non-treponemal tests must be confirmed by Treponemal tests using
specific T: pallidum antigens to rule out the biological false positive reactions.
1. T. pallidum Immobilization (uses live T.pallidum)
TPI test is based on the ability of patient's antibody and complement to
immobilize the live actively motile T.pallidum (Nichols strain), observed under
dark ground microscope.
1. Fluorescent Treponemal Antibody-Absorption Test (Uses killed T.pallidum)
It is an indirect immunofluorescence test. Smears of killed T.pallidum (Nichols
strain) are prepared on slides and fixed. The patients serum is allowed to react
with the smear. The antibodies that bind to the fixed smear are detected by
treating the smear with fluorescent labelled antihuman immunoglobulin. In a
positive test, Treponemes fluoresce.

20. 3. T. pallidum Hemagglutination Assay (TPHA)
Patient's serum pretreated with Reiter treponemes is added to a drop of tanned
sheep RBCs coated with T: pallidum antigens. In a positive reaction , Smooth mat of
agglutinated cells is formed.
TPHA is affordable, easy to perform, available as commercial kit and no special
equipment is needed.
Sensitivity and specificity of TPHA are excellent in all the stages, except for primary
syphilis where die sensitivity is low.

21. Enzyme lmmunoassays
ELISA specific to IgG and IgM have been developed for the diagnosis of syphilis.
They have excellent sensitivity and specificity.
Wesrern Blot
Western blot is available for detecting lgG and IgM antibodies separately.
It is highly sensitive and specific.
Molecular Methods
PCR-based techniques are available to amplify T. Pallidum specific genes. PCR is of
paramount importance in the diagnosis of congenital and neurosyphilis.

22. Treatment
Pencillin is the drug of choice for all the stages of syphilis.
In Primary, secondary, or early latent syphilis: single dose of Penicillin G is given.
Alternate drug used is tetracycline.
Prophylaxis
Prophylactic use of sex barriers (condom).

23. NON-VENEREAL TREPONEMATOSES
● Yaws is an endemic disease caused by T.pallidum subspecies pertenue.
Manifested as extra genital papule on extremities.Destructive lesions of bones
are common.
● Pinta is a skin disease in which papule appears which does not ulcerate and
become lichenoid or psoriform patch. It is caused by Treponema carateum.
● Endemic syphilis is caused by T.pallidum subspecies endemicum. It is
transmitted non- venerally. It usually presents with mucosal patches and
eruptions.

24. BORRELIA
It is large, motile spirochete with 3-10 irregular wide coils.
They can be stained using ordinary dye and are gram negative.
Pathogenic Borrelia species are :
● B. recurrentis - causing relapsing fever.
● B. vincentii causing Vincent’s angina.
● B. burgdoferi causing Lyme disease.

26. B. burgdorferi
It is large, motile spirochete with 3-10 irregular wide coils.
It is a microaerophile.
It causes Lyme disease.
Lyme disease is transmitted by tick bite (lxodes ricinus complex).
Rodents and deer are main reservoirs of Lyme disease.

27. Clinical Manifestations
Lyme disease occurs through four stages:
Stage 1: After an incubation period of 3-32 days, an annular maculopapular lesion
develops at the site of the tick bite called erythema migrans, commonly involving
thigh, groin, and axill.
Stage 2: Early disseminated infection. B. burgdoiferi spread hematogenously to
many sites within days or weeks resulting in:
● Secondary annular skin lesions
● Musculoskeletal pain (arthralgia)
● Profound malaise and fatigue
● Neurological abnormalities, like meningitis, encephalitis etc
● Cardiac involvement, including atriovencricular block.

28. Stage 3: Late persistent infection : About 60% of untreated patients develop frank
arthritis involving large joints (especially the knees), lasting for weeks or months in a
given joint. Some cases of Lyme arthritis are refractory for treatment.
Post-Lyme syndrome (Chronic): Few patients present with chronic fatigue
symptoms and neurocognitive manifestations, develop after months to years of
infection.

30. Laboratory Diagnosis
Culture
It is very time consuming and hence not routinely used. BSK medium is used for culture.
Cultures are incubated and examined under dark field microscopy weekly for two
months.
Serology
Serological tests such as ELISA and immunofluorescence (IF) have been described and
immuno blotting (Western blot) recommended for confirmation.
Molecular method
PCR is superior in detection of B. Burgdorferi in joint fluid.

31. Treatment
Oral doxycycline is the drug of choice, except for children where amoxicillin is
given.
For CNS or CVS infection: Cefrtiaxone is given for 14-28 days

32. B. recurrentis
They are motile, gram negative and possess 5-8 irregular spirals at intervals of 2 micrometre with
pointed end.
It stains well with geimssa or wright stain.
It causes epidemic relapsing fever characterized by recurrent episodes of fever and nonspecific
symptoms.
It is transmitted by human body louse (pediculuc humanus). Borreliae are introduced by crushing of
the louse (e.g. by scratching) leading to deposition of insect's infected hemolymph containing
numerous spirochetes on the abraded skin and mucous membranes.
Another type of relapsing fever, endemic relapsing fever is caused by B. duttoni, B. hermsii and B.
turicatae. It is transmitted by the bite of an infected tick(Ornithodoros species).

33. Pathogenesis
From the inoculated site, Borrelia spreads rapidly leading to bacteremia and fever.
However, the borrelial surface antigens frequenly undergo antigenic variation.
Each time, new antigens are produced which can evade host's immwie system
leading to repeated bacteremia and recurrent febrile episodes.
Incubation period is about 7-8 days.
Recurrent febrile episodes lasting for 3-5 days occur intervening with afebrile
periods of 7-9 day. Subsequent episodes are shorter.
It may preset like alteration of sensorium, abdominal pain, vomiting and diarrhea
Splenomegaly is common.

34. Laboratory Diagnosis
Microscopy
Giemsa-stain : gram negative spirochete with 5-8 irregular spirals at regular
intervals.
Dark ground microscope - Borrelia can be identified by their active movements.
Culture: the confirmation is made by isolation of Borrelia from blood. But is time
consuming and hence not routinely used.
ELISA and IFA (indirect fluorescence assay) are available to detect serum antibodies.

36. Treatment
Antibiotics such as doxycycline or erythromycin are the drug of choice for relapsing
fever.
Recommended schedule is single dose for epidemic RF, and 7-10 days course for
endemic RF.

37. LEPTOSPIRA INTERROGANS

38. LEPTOSPIRA INTERROGANS
They are actively motile spirochetes possessing numerous closely wound spirals and
characteristic hooked ends.
They possess a single endo flagellum attached at the pole.
It is the causative organism of Leptospirosis, a zoonotic disease.
They do not stain readily but may be observed by dark ground microscopy.
L. interrogans comprises of 25 serogroups which further consist of over 250 serovars.

39. Pathogenesis
Leptospirosis is zoonoti disease.
Direct human-to-human transmission does not occur.
It is transmited by:
● lndirecl contact with water, moist soil and wet surfaces contaminated with
animal urine or
● Direct contact with infected urine
important sources of infection are rats, dogs, cattle and pigs.

40. After entering through the mucosa ( conjunctival or oral) or abraded skin,
L.interrogans spill over to the bloodstream and then disseminate hematogenously to
various organs including brain, liver, lung, heart and kidney.
During the acute phase of the disease, leptospires are seen in the blood but can
seldom be demonstrated after 8-10 days.
The pathogenesis of leptospirosis is assumed to be a vasculitis resulting in damage
to the endothelial cells of small blood vessels.

41. Clinical Manifestations
The incubation period is usually about 10 days (range 2-26 days).
The onset of clinical illness is usually abrupt, with nonspecific, influenza-like
constitutional symptoms such as fever, chills, headache, severe myalgia, and malaise.
Severe systemic disease (Weil’s disease) includes renal failure, hepatic failure, and
intravascular disease, and may result in death.
Leptospirosis is an established cause of aseptic meningitis.

42. Lab Diagnosis
Microscopy
Leptospires disappear from the blood after the first week, blood examination is
helpful only in the early stages of the disease.
Leptospires may be found in the urine during the second week and intermittently for
4-6 weeks or even longer.
They do not stain by ordinary stain, but can be stained by sliver impregnation stains
such as Fontana stain and modified Steiner technique. They are seen as tightly and
regularly coiled, with characteristic hooked ends.
They may also be observed under dark ground microscopy.

44. Culture
Leptospira is obligate aerobe and is highly fastidious, requires enriched media.
Specimen of blood during first week and urine in second week can be used.
Korthof's media with rabbit blood and Fletcher's semisolid media can be used.
EMJH medium (Ellinghausen, McCullough, Johns01i, Harris) is a semisynthetic
liquid medium, most commonly used nowadays.
Isolation of Leptospira confirms the diagnosis but Culture technique is laborious,
technically demanding and time-consuming.

45. Serology for antibody detection:
Antibodies begin to appear at the end of first week and continue to rise till the
fourth week.
A rapid dip-stick assay has been developed for detection of leptospira specific IgM
antibody.
Other testes include ELISA, complement fixation test, haemagglutination test and
indirect immunofluorescence.

46. Treatment
Mild leptospirosis should be treated with oral doxycycline (100 mg twice a day for 7 days).
Amoxicillin can be given alternatively.
Severe leptospirosis: Penicillin is the drug of choice, alternatives being ceftriaxone or cefotaxime.
Control Measures lnclude
● Chemoprophylaxis with doxycycline is recommended for anticipated short-term exposures,
such as military training or travelling or fresh-water swimming.
● General sanitation approaches including proper waste disposal.
● Rodent control.
● Avoidance of swimming in contaminated places.
● Health education.

47. Thank You