SPIROCHETES-I

1. SPIROCHETES-I Dr. Mohit Bhatia Assistant Professor Department of Microbiology AIIMS, Rishikesh

2. Spirochetes • Thin, flexible, elongated spirally coiled helical bacilli • Speira = coil • Chaite = hair

3. Classification of Spirochetes • Class Spirochaetaceae • Spirocheta • Cristispira • Treponema • Borrelia • Class Leptospiraceae • Leptospira • Leptonema.

4. Ultrastructure of Spirochetes • Outer membrane • ™Periplasmic space containing flagella • ™Peptidoglycan layer • ™Inner (cytoplasmic) membrane • Motile with endoflagella

5. Morphological differences between Treponema,Borrelia and Leptospira

6. Spirochetes - Classification

7. TREPONEMA

8. TREPONEMA • Pathogenic species • T. pallidum subspecies pallidum • T. pallidum subspecies pertenue • T. pallidum subspecies endemicum • T. carateum • Almost identical in their morphology, antigenic structure and in genetic composition

9. TREPONEMA PALLIDUM • ™Morphology: extremely thin and delicate with tapering ends • ™Size: 6–14 μm × 0.2 μm • ™Spirals: 6–12 spirals at intervals of 1 μm • ™Motility: flexion extension, translatory and corkscrew motility • ™Endoflagella: About 3–4 flagella - motility & highly antigenic

10. Microscopy & Culture • Dark ground or phase contrast microscope • ™Staining: Do not take up Gram stain • Fluorescence staining • Sliver impregnation methods • ™Cultivation: Pathogenic treponemes cannot be grown in artificial culture media. Maintained in rabbit testes. E.g, „Nichols strain • Non-pathogenic Treponemes – grow in Smith Noguchi medium under strict anaerobic conditions. E.g: Reiter’s strain & Noguchi strain

11. Antigens Group-specific antigen: Protein antigen • Present in all treponemes • Antibodies detected using antigens of Reiter treponemes Species-specific antigen: Polysaccharide • Antibodies detected by using specific T. pallidum antigens Non-specific antigen: Heterophile antigen - Antibody detected using beef heart antigen

12. PATHOGENESIS OF SYPHILIS • Mode of transmission: • Venereal • Non-venereal - direct contact, blood transfusion or transplacental • ™Spread: T. pallidum penetrates through mucosa or abraded skin  Enter lymphatics and blood  systemic  primary lesion • ™Incubation period: Variable (9–90 days) Inversely proportional to the number of organisms inoculated

13. PRIMARY SYPHILISNIFESTATIONS OF SYPHILIS • Primary (or hard) chancre: • Single painless papule ulcerated & indurated • Covered by thick exudate rich in spirochetes • Common sites – penis, cervix or labia • Heals within 4–6 weeks

14. CLINICAL MANIFESTATIONS OF SYPHILIS • ™Regional (usually inguinal) lymphadenopathy • Painless firm, non-suppurative, and often bilateral • May persist for months • ™™If acquired by non-venereal mode: - „Direct contact → extragenital, usually on the fingers - „Blood transfusion → primary chancre does not occur

15. SECONDARY SYPHILIS • Develops 4–8 weeks after healing of primary lesion • Skin and mucous membranes - commonly affected • ™Skin rashes

16. Secondary Syphilis • ™Condylomatalata: Mucocutaneous papules coalesce to form large pink to grey lesion in warm moist intertriginous areas (such as perianal region, vulva, and scrotum) • ™Mucous patches (superficial mucosal erosions)

17. Latent Syphilis • Absence of clinical manifestations of syphilis with positive serological tests and normal CSF findings. • ™Patients are still infectious - bloodstream or in utero • ™Fates: • Persistent lifelong infection (common) • Development of late syphilis (rare) • „Spontaneous cure

18. Late or Tertiary Syphilis • Gumma (late benign syphilis): Locally destructive granulomatous lesions - bone and skin • ™Neurosyphilis: „- Meningeal syphilis (meningitis) - Meningovascular syphilis (vasculitis of arteries embolic stroke) - „General paresis of insane „- Tabes dorsalis • Cardiovascular syphilis: aneurysm of ascending aorta and aortic regurgitation

19. Congenital Syphilis • Transmission - at any stage of pregnancy • Fetal damage - after fourth month of gestation Manifestations of congenital syphilis include: • ‰Earliest manifestations : (within 2 years of age) & infectious - Snuffles, mucocutaneous & bone changes, hepatosplenomegaly • ‰Late congenital syphilis: (after 2 years) & non-infectious • Interstitial keratitis, eighth-nerve deafness, bilateral knee effusions • Residual stigmata - Hutchinson’s teeth (notched central incisors), Mulberry molars, Saddle nose, and saber shins

20. LABORATORY DIAGNOSIS OF SYPHILIS • Direct Microscopy • Dark Ground Microscopy (DGM) • Slender, flexible, spirally coiled bacilli with tapering ends • Motility: Flexion-extension type, corkscrew motility, Soft bending at right angle to the midpoint • Sensitivity of DGM - 80% • Detection limit of 104 bacilli/mL

21. LABORATORY DIAGNOSIS OF SYPHILIS • Direct Fluorescent Antibody Staining - Distinct, sharply outlined, apple green fluorescent bacilli • ™Sensitivity - 100% when smear made from fresh lesions are examined

22. LABORATORY DIAGNOSIS OF SYPHILIS • Silver Impregnation Staining • Increase thickness - Levaditi stain -tissue section - Fontana stain – exudate smears • Cultivation- maintained by subcultures in rabbit testes

23. Serology (Antibody Detection) • 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 • Group-specific tests: Detect group or genus-specific antibody by using Reiter treponemal strains possessing protein antigen present in all treponemes.

24. Standard Tests for Syphilis • Non-treponemal or Non-specific tests or STS (Standard Tests for Syphilis) • Detect reagin antibody using cardiolipin antigen extracted from beef heart • Cardiolipin antigen - diphosphatidyl glycerol • Reagin antibodies are IgG or rarely IgM type • Slide flocculation tests - VDRL, RPR, USR, TRUST • Wassermann test (e.g. of complement fixation test) • Kahn test (e.g. of tube flocculation test

25. Venereal Disease Research Laboratory (VDRL) • ‰Widely used, simple and rapid serological test • VDRL antigen - cardiolipin antigen to which cholesterol and lecithin are added • Slide Flocculation test

26. Venereal Disease Research Laboratory (VDRL) • Qualitative test: Inactivated serum + a drop of VDRL antigen rotated at 180 rpm for 4 minutes in a VDRL rotator  examined under microscope - Non-reactive: Uniformly distributed fusiform crystals • Reactive: Medium to large clumps • ‰Quantitative test: Test performed with serial dilutions (1:2, 1:4, 1:8 and so on) of serum done with 0.9% saline • ‰VDRL-CSF: No preheating of CSF is needed

27. VDRL TEST Positive Negative

28. VDRL v/s RPR

29. VDRL v/s RPR

30. Other Reagin Antibody Tests • Unheated Serum Reagin Test (USR) is similar to VDRL except for: • EDTA - antigen stabilizer  daily preparation of antigen is eliminated • Choline chloride - inhibit the non-specific inhibitors in serum  pre-heating of serum is not needed • Toluidine Red Unheated Serum Test (TRUST) • Modified RPR test where toluidine red pigment particles • Does not require microscope for examination

31. Advantages of Non-treponemal Tests • ™To monitor the response to treatment • Reagin tests usually become negative 6–18 months after the effective treatment • ™Neurosyphilis: VDRL detects CSF antibodies • ™Detectable 7–10 days after the appearance of primary chancre (or 3–5 weeks after acquiring the infection) • Sensitivity: Varies from 78 to 85% in primary stage, 100% in secondary stage and 95–98% in latent stage

32. Disadvantages of Non-treponemal Tests • Biological false-positive (BFP) reactions: Positive non-treponemal tests, with negative treponemal tests, in absence of syphilis and no technical faults. BFP Antibodies - 1% of normal sera, IgM type • Conditions - lepromatous leprosy, relapsing fever, malaria, tropical pulmonary eosinophilia, viral hepatitis, infectious mononucleosis, HIV, pregnancy and IV drug abusers • ™Prozone phenomena • ™Sensitivity of non-treponemal tests is low in late stage • ™Non-treponemal tests are used as screening tests

33. BIOLOGICAL FALSE POSITIVE(BFP) REACTIONS • As cardiolipin Ag is present both in T.pallidum and in mammalian tissues, reagin Abs may be induced by treponemal or host tissue antigens which accounts for BIOLOGICAL FALSE POSITIVE(BFP) • BFP defined as “positive reactions” obtained in cardiolipin tests, with negative results in specific treponemal tests, in the absence of past or present treponemal infections and not by technical faults • They represent non-treponemalcardiolipin antibody responses

34. Represent non treponemal cardiolipin Ab responses • BFP reactions-may occur in about 1% of normal sera • Clinically BFP reactions classified as acute & chronic • Acute BFP-last only for weeks or months, associated ē acute infections, injuries or inflammatory conditions • Chronic BFP-persist longer than 6 months, seen in SLE & other collagen diseases • Other conditions associated ē BFP reactions are leprosy, malaria, relapsing fever, infectious mononucleosis, hepatitis , tropical eosinophilia , pregnancy and menstruation.

35. Syphilis Tests - Sensitivity & Specificity

36. Syphilis Tests - Sensitivity & Specificity

37. Treponemal or Specific Tests • T. pallidum Immobilization (TPI) test • Principle: patient’s antibody and complement to immobilize the live actively motile T.pallidum (Nichols strain) - observed under dark ground microscope

38. Treponemal or Specific Tests Fluorescent Treponemal Antibody-Absorption Test (FTA -ABS) • Uses killed T.pallidum, indirect fluorescent antibody technique • Patient’s serum diluted with an extract of non-pathogenic Reiter treponemes to remove group specific treponemal antibodies • Layered on a slide previously coated with killed T. pallidum • Serum antibodies bound to T. pallidum can be detected by addition of fluorescent labeled anti-human immunoglobulin • Examined under fluorescent microscope

39. Treponemal or Specific Tests • ™Advantages: Highly sensitive and specific in all stages of syphilis • First serological test to be positive following infection • Detects CSF antibodies • ™Disadvantage: False positive results in Lyme disease

40. Treponemal or Specific Tests • T. pallidum Hemagglutination Assay (TPHA) • Tanned sheep RBCs coated with T.pallidumantigens • Reactive result: Smooth mat of agglutinated cells in microtiter plate • Nonreactive result: Compact button in the center of the well • ™Quantitation- done by serial dilution of patient’s sera • Advantages: Affordable, easy to perform,available as commercial kit and no special equipment is needed, detects CSF antibodies - Sensitivity and specificity of TPHA are excellent

41. TPHA

42. Treponemal or Specific Tests • Enzyme Immunoassays • ELISA specific to IgG and IgM, ™They have excellent sensitivity and specificity • Western Blot • Detects IgG and IgM antibodies separately, highly sensitive and specific • Group-specific Test - Reiter’s protein complement fixation test (RP-CFT) • Molecular Methods - PCR-based techniques

43. Diagnosis of congenital syphilis • Definitive diagnosis: Demonstration of T. pallidum by DGM of umbilical cord, placenta, nasal discharge, or skin lesion material • Presumptive diagnosis: • Infant born to a mother who had syphilis at the time of delivery regardless of findings in the infant • Reactive treponemal test in infant

44. Diagnosis of congenital syphilis • ‰One of the following additional criteria: • Clinical signs/symptoms of congenital syphilis • Abnormal CSF findings without other cause • Reactive VDRL-CSF test • Reactive IgM antibody test specific for syphilis (IgM FTA ABS or IgM ELISA). • Presence of specific IgM in neonatal serum confirms the diagnosis

45. DIAGNOSIS OF NEUROSYPHILIS CSF cell counts of 5cells/cu.mm Protein values above 40mg/100ml VDRL(NOT RELIABLE );Non reactive in 30-60% Negative TPHA of CSF excludes neurosyphilis Positive TPHA or FTA-ABS of CSF does not necessarily indicate active diseasesince reactivity may be caused by transudation of immunoglobulins from serum into CSF. TPHA index is a reliable parameter The TPHA index, which relates the CSF-TPHA titre to the albumin quotient and thus excludes errors from disturbed function of the blood-brain barrier,

46. Syphilis and HIV • Both syphilis and HIV affect each other’s pathogenesis • Problems in the diagnosis of syphilis in HIV infected people are: • Confusing clinical picture, Lack of serologic response • Unusually high titers in non-treponemal tests • Failure of non-treponemal test titers to decline even after treatment • Disappearance of treponemal test reactivity over time

47. Treatment Syphilis • ‰Penicillin is the drug of choice for all the stages of syphilis • Primary, secondary, or early latent syphilis: single dose of Penicillin G • Late latent CVS or benign tertiary stage: penicillin G is given single dose weekly for 3 weeks • ‰Alternative drug is used in patients with penicillin allergy: • Primary, secondary, latent, CVS or benign tertiary syphilis—tetracycline • Neurosyphilis or pregnancy or associated HIV—desensitization to penicillin

48. Evaluation after Treatment • Non-treponemal tests • ™For primary and secondary syphilis: at least fourfold decline in the titer by the third or fourth month and an eightfold decline in the titer by sixth to eighth month • ™Latent or late syphilis, or patients with multiple episodes of syphilis: gradual decline in titer, low titers may persist for years

49. NON-VENEREAL TREPONEMATOSES

50. NON-VENEREAL TREPONEMATOSES