SPIROCHETESI Dr Mohit Bhatia Assistant Professor Department of

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

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

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

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

Morphological differences between Treponema, Borrelia and Leptospira Treponema Borrelia Leptospira Size 6 -14μm X 0. 2μm 10 -30μm X 6 -20 μm X 0. 2 -0. 5μm 0. 1μm Spirals 6 -12 in no. 3 -10 in no. Numerous and tightly coiled with hooked ends Wave length 1μm 3μm Amplitude of spiral Endoflagella at each pole 1 -1. 5μm Up to 2μm 0. 1μm 3 -4 7 - 11 0. 5μm 1

Spirochetes - Classification Spirochaetes Disease Transmission T. pallidum Syphilis Sexual T. pertenue Yaws T. endemicum Endemic syphilis T. carateum Pinta B. recurrentis Relapsing fever (epidemic) Direct contact Louse borne B. duttonii, B. hermsii B. burgdorferi Relapsing fever (endemic) Tick borne Lyme disease Tick borne L. interrogans Leptospirosis 1. Milder form 2. Severe form (Weil’s disease) Contact with rodent urine

TREPONEMA

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

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

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

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

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

PRIMARY SYPHILIS NIFESTATIONS 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

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

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

Secondary Syphilis • �Condylomata lata: 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)

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

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

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

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/m. L

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

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

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 genusspecific antibody by using Reiter treponemal strains possessing protein antigen present in all treponemes.

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 Ig. G or rarely Ig. M type - Slide flocculation tests - VDRL, RPR, USR, TRUST - Wassermann test (e. g. of complement fixation test) - Kahn test (e. g. of tube flocculation test

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

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

VDRL TEST Positive Negative

VDRL v/s RPR VDRL Results read microscopically clumps are smaller RPR Results read macroscopically Finely divided carbon particles coated cardiolipin antigens are used so that larger visible clumps are formed Antigen, once reconstituted, EDTA is used as stabilizer; hence should be used within 24 hours RPR antigen can be stored longer (up to 6 months at 4100 C) Preheating of serum is required Preheating of serum is not to remove non specific required as choline chloride is inhibitors used to remove inhibitors

VDRL v/s RPR VDRL RPR Blood, plasma, serum, and CSF can be tested Blood, plasma and serum can be tested but not CSF Rotation of slide is done for 4 mins Sensitivity in primary syphilis is 78% It is cheaper; one vial of VDRL antigen can be used for 250 tests. It is preferred for field studies and for antenatal screening Rotation of card is done for 8 mins Sensitivity in primary syphilis is 86% RPR is expensive than VDRL. It is preferred when sample load is less.

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

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

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, Ig. M 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

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-treponemal cardiolipin antibody responses

q. Represent non treponemal cardiolipin Ab responses q. BFP reactions-may occur in about 1% of normal sera q. Clinically BFP reactions classified as acute & chronic q. Acute BFP-last only for weeks or months, associated ē acute infections, injuries or inflammatory conditions q. 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.

Syphilis Tests - Sensitivity & Specificity Sensitivity (%) Treponemal Primary Secondary Latent Late Specificity (%) FTA-ABS 84 100 96 97 TPPA 88 100 ? 98 TPHA 76 100 97 94 99 EIA 90 100 ? 99 Western blot 90 100 ? 98 TRUST 85 100 98 ? 99

Syphilis Tests - Sensitivity & Specificity Sensitivity (%) Primary Secondary Latent Late Specificity (%) Non treponemal VDRL 78 100 95 71 98 RPR 86 100 98 73 98 USR 80 100 95 ? 99 TRUST 85 100 98 ? 99

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

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

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

Treponemal or Specific Tests • T. pallidum Hemagglutination Assay (TPHA) - Tanned sheep RBCs coated with T. pallidum antigens - 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

TPHA

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

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

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 Ig. M antibody test specific for syphilis (Ig. M FTA ABS or Ig. M ELISA). • Presence of specific Ig. M in neonatal serum confirms the diagnosis • -

DIAGNOSIS OF NEUROSYPHILIS CSF cell counts of 5 cells/cu. mm Protein values above 40 mg/100 ml 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 disease since 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,

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

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

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

NON-VENEREAL TREPONEMATOSES

NON-VENEREAL TREPONEMATOSES Feature Venereal Syphilis Yaws Endemic Syphilis Agent T. pallidum T. pertenue T. endemicum T. carateum Mode of transmission Sexual, Skin-to-skin Transplacental Blood Household contacts: kissing, sharing utensils or insect vector Skin-to-Skin Age Adulthood Early childhood Late childhood Early childhood Pinta

NON-VENEREAL TREPONEMATOSES Feature Venereal Syphilis Yaws Endemic Syphilis Pinta Primary lesion Chancrepainless nonindurated Lymphadenop athy Papilloma, Rarely seen often ulcerative Lymphadenop athy Non ulcerating pruritic papule Site of lesion Extremities, face Genital, oral, anal Oral

NON-VENEREAL TREPONEMATOSES Feature Venereal Syphilis Yaws Endemic Syphilis Pinta Secondary lesions Skin rashes Mucosal patches Condylomata lata Skin lesionsmacular or papular Periostitis Oral mucous Pintides, patches Pigmented & Periostitis, pruritic Lymphadenop athy Relapses 25% Common Unknown None

NON-VENEREAL TREPONEMATOSES Feature Venereal Syphilis Yaws Endemic Syphilis Late complications Gummas, CVS and CNS lesion Destructive gummas of skin, bone, cartilage Destruction of the nose, maxilla, palate, and pharynx is termed as gangosa Pinta Non destructive, dyschromic macule

YAWS

Lesions of Pinta