immunology test 2

commensalism

• neither organism is significantly helped or harmed

mutualism

• both the host and microorganism benefit

parasitism

• the microorganism benefits while harming the host

symbiosis

• host and microorganism live together long term

  • normal biota DOES NOT = infection

infectivity

• ability to establish an infection

• can it infect?

pathogenicity

• ability to cause disease

• can it cause disease?

virulence

• extent of damage/pathology produced

• how severe is the damage?

knowledge check: a factor that helps a bacterium evade phagocytosis and cause more severe disease is a…

• virulence factor

bacterial virulence factors

• capsule - resists phagocytosis/ immune interactions

• pili/fimbriae - attachment to host cells

• flagella - motility; may aid adherence

• endotoxin (LPS) - strong inflammatory cytokine release

• exotoxins - secreted proteins that damage/ alter host cells

bacterial virulence factors - immunology connection

• endotoxin → cytokine release

• capsule → phagocytosis/complement

how does the immune system respond to EXTRACELLULAR BACTERIA?

• antibodies

• complement

• phagocytosis

• inflammatory cytokines

how does the immune system respond to INTRACELLULAR BACTERIA?

• cell-mediated immunity

• CD4+ T-cell cytokine responses

• cytotoxic T cell responses

*type of immune response depends partly on where the organism is located

bacteria can evade host defenses

• prevent effective chemotaxis

• interfere with phagocyte attachment

• resist destruction after phagocytosis

• interfere with complement

• degrade IgA

• alter or mask surface antigens

*host response and laboratory findings may VARY during infection

ask FIRST before choosing laboratory detection method:

• are we detecting the pathogen…

OR

• are we detecting evidence of the host response?

what can the laboratory detect?

• living organism → culture

• morphology → microscopy/staining

• bacterial antigen → immunologic antigen detection

• DNA or RNA → molecular testing

• patient antibody → serology

• bacterial proteins → proteomic methods (gene sequence)

direct detection

• “can i detect the PATHOGEN or one of its components?”

• culture → organism

• antigen assay → bacterial antigen

• PCR → bacterial nucleic acid

serology

• “has the patient produced ANTIBODIES against the organism?”

• detects the HOST immune response

• does NOT directly detect the bacterium

*Positive antibody DOES NOT equal direct detection of the organism

why detect antibody instead of the organism?

• difficult to culture: some organisms are difficult to recover in routine culture

• hazardous to culture: serology may provide safer indirect evidence

• ORGANISM MAY BE GONE: complications can appear after the original infection has resolved

• evidence of exposure: antibody can document a host immune response

*TRADEOFF: antibody production takes time

timing matters in serologic testing

• exposure/infection → early infection (pathogen may be present/antibody undetectable) → immune response (antibody becomes detectable) → convalescence (antibody may persist)

• serologic results must be interpreted in relation to TIME

• early negative antibody result DOES NOT mean we rule out infection

IgM and IgG think pattern (not just pos/neg)

• IgM: often appears earlier, may support RECENT exposure/infection

• IgG: usually develops LATER, may persist AFTER INFECTION, may reflect PAST exposure or a LATER immune response

• IgM = recent & IgG = past is NOT a universal rule

sometimes one specimen is not enough

• acute specimen: collected EARLY in the illness

• convalescent specimen: collected later, after the immune response has had time to develop

• COMPARE ANTIBODY LEVELS - look for a significant change in atnibody titer (usually 4-fold)

what is a fourfold rise in titer?

• Acute - 1:40 → 1:160 in convalescent

  • 40 × 4 = 160 → fourfold rise

• 1:40 → 1:80 is NOT fourfold, instead is twofold

  • usually not considered significant

why can serology be misleading?

• too early: antibody not yet detectable

• cross reactivity: antibody reacts with related antigens

• vaccination: may affect some antibody results

• past exposure: antibody may persist

• immunosuppression: weak or absent antibody response

• single specimen: may not show when exposure occurred

*bottom line = interpret the result in context

what are febrile agglutinins?

• PATIENT ANTIBODIES that react with standardized bacterial antigens

• a reactive antigen—antibody rxn produces visible agglutination

why were/are febrile agglutinins used?

• relatively rapid and simple serologic testing

• historically useful when culture was difficult/slow/hazardous

• still performed in some clinical laboratories

important limitations of febrile agglutinins

• antibodies take time to develop

• cross reactivity may cause false positives

• a single reactive result does not prove active infection

• more specific testing is preferred when available

common febrile agglutinin tests

• Widal test for Salmonella

  • O (somatic) and H (flagellar) antigens used

  • detects antibodies reacting with salmonella antigens

• Weil-Felix test for Rickettsial disease

  • Proteus antigens used: OX19, OX2, OXK

  • detects cross reacting antibodies associated w some rickettsial infections

• Huddleston test for Brucella

  • Brucella antigen used

  • detects antibodies reacting with brucella

• Francisella tularensis test

  • F. tularensis antigen used

  • detects antibodies reacting with F. tularensis

knowledge check: a patient is tested for antibodies to a bacterial pathogen very early in the illness. the antibody test is negative. why can this be?

• the patient may not yet have produces detectable antibody

4 infections = 4 diagnostic problems

• Group A Strep (GAS): the organism may be gone when immune mediated sequelae appear → detect antibody to a previous infection

• Mycoplasma pneumoniae: specific antibody and nonspecific cold antibodies can coexist → know what the antibody is actually binding

• Rocky Mountain Spotted Fever (RMSF): the patient may be very ill before antibodies become detectable → timing can make serology negative

• Tuberculosis: antibody is not the useful immune marker → detect T cell sensitization/ IFN-gamma response

Group A streptococcus - PRIMARY infection

• pharyngitis or impetigo/pyoderma

• M protein

  • major virulence factor

  • inhibits phagocytosis

  • limits C3 deposition (innate immune)

• lipoteichoic acid + Protein F

  • allow for attachment in skin or pharyngeal area

• SpeA/SpeC

  • superantigens

• SpeB

  • tissue invasion/injury - destroy host cell

Group A Strep - immune targets

• extracellular products can stimulate antibodies

  • streptolysin O

  • DNase B

  • hyaluronidase

  • streptokinase

  • NADase

• these antibody responses become useful AFTER the primary infection

GAS: what can the immune system recognize?

GAS: primary infection vs immune mediated sequences

1. GAS infection: pharyngitis or impetigo/pyoderma

2. immune response: antibodies and cellular responses develop

3. infection resolves: GAS may no longer be detectable at the original site

4. later: sequelae

• acute rheumatic fever

  • cross reactive immune response → inflammation of joints and heart → possible chronic VALVULAR DAMAGE/ RHEUMATIC HEART DISEASE

• poststreptococcal glomerulonephritis

  • immune complex deposition + complement activation in glomeruli → hematuria, proteinuria, edema, hypertension, decreased GFR

acute rheumatic fever (molecular mimicry)

• Previous GAS (esp. after pharyngeal infection) → immune response (antibodies/cellular responses to GAS antigens) → cross-reactivity (SOME GAS EPITOPES RESEMBLE HOST TISSUE ANTIGENS)

• molecular mimicry leads to inflammatory joint pain, carditis, possible rheumatic heart disease/valvular damage

post strep glomerulonephritis: immune complex disease

• previous GAS (pharyngitis/skin) → antigen + antibody (immune complexes form) → glomeruli (immune complexes DEPOSIT) → inflammation (complement activation + tissue injury)

• TYPE III HYPERSENSITIVITY

  • pt experiences: hematuria, proteinuria, edema hypertension, decreased GFR

ASO vs Anti-DNase B (site of infection matters)

• ASO

  • target: streptolysin O

    • commonly elevated after PHARYNGITIS

  • peak response: 3-6 wks

  • post BOC association: recent GAS, especially THROAT

• Anti-DNase B

  • target: DNase B

    • commonly elevated after pharyngitis

    • especially useful after PYODERMA/IMPETIGO

  • peak response: 6-8 wks

  • best BOC association: recent GAS after SKIN INFECTION/POST STREP GLOMERULONEPHRITIS

ASO testing - rapid slide

• latex agglutination

• streptolysin O antigen is attached to latex particles

• patient ASO cross-links particles → VISIBLE AGGLUTINATION

• rapid, simple, affordable, still used in clinical labs

ASO testing - automated methods

• nephelometry → measures scattered light

• immunoturbidimetry → measures decreased transmitted light

• provides QUANTITATIVE ASO results

both ASO testing methods…

• same antigen-antibody rxn

• different ways of detecting immune complexes

• IMPORTANT DISTINCTION: ASO latex agglutination detects ASO specifically

historical note: streptozyme

• what it was: qualitative screening agglutination assay designed to detect one or more anti-strep antibodies ( ASO, Anti-DNase B, Anti-hyaluronidase, Anti-streptokinase, Anti-NADase)

• how to teach it now:

  • NO LONGER ROUTINELY AVAILABLE/MANUFACTURED

knowledge check: a child develops periorbital edema, dark urine, and hypertension 3 wks after impetigo. which result best supports recent GAS infection?

• elevated anti-DNase B

  • IMPETIGO = main clue

  • skin GAS may produce little ASO, Anti-DNase B is especially useful after pyoderma and supports PSGN history

Mycoplasma pneumoniae - what makes it different?

• very small bacterium

• NO CELL WALL

• respiratory infection

• gradual onset

• persistent cough

• fever, headache, malaise

• often called “WALKING PNEUMONIA”

Mycoplasma pneumoniae - what happens immunologically ?

• infection stimulates:

  • M. pneumoniae-specific antibodies

  • cytokine/chemokine response

  • antibodies, including COLD AGGLUTININS

Mycoplasma pneumoniae - possible extrapulmonary effects

• stevens-johnson syndrome

• raynaud phenomenon

• COLD AGGLUTININ-ASSOCIATED RBC AGGLUTINATION/HEMOLYSIS

• the immune response can extend beyond the lungs

M. pneumoniae - specific antibody response

• infection → patient makes antibodies against M. pneumoniae antigens → specific IgM/IgG

• these antibodies support organism specific serology

M. pneumoniae NONSPECIFIC cold agglutinin

• infection → IgM cold agglutinins develop → IgM binds RBC I antigen → RBC agglutination at colder temps

• NOT M. pneumoniae specific

what is happening in cold agglutinins?

• antibody: usually IgM, directed against RBC I antigen, pentameric IgM efficiently bridges RBCs and leads to strong agglutination

• RBC effect: IgM binds RBCs best at COOLER temps, visible RBC agglutination can occur

• if clinically significant: complement can be activated and lead to RBC destruction + hemolytic anemia

are cold agglutinins good for diagnosing mycoplasma?

• NOT BY THEMSELVES

  • why not?

    • cold agglutinins develop in only abt HALF of pts

    • NOT SPECIFIC for M. pneumoniae

• better approach:

  • M. pneumoniae specific IgM/IgG serology

  • paired specimens when serology is used

  • molecular testing for current infection

mycoplasma serology: IgM vs IgG

• specific IgM = supports current/recent infection

• IgG alone = may reflect previous exposure; adults may show IgG on reinfection

• rising IgG in paired sera = supports recent infection

• early negative antibody = does NOT exclude infection

rocky mountain spotted fever - what happens?

• R. rickettsii

  • tick borne, obligate intracellular bacterium

    • replicates INTRACELLULARLY

  • targets vascular endothelial cells

    • endothelial injury increases vascular permeability

• clinical effects

  • leaky vessels can lead to:

    • edema

    • hypovolemia (losing fluid to extracellular space)

    • hypotension

  • overall picture:

    • fever

    • severe headache

    • GI symptoms

    • rash

    • potentially severe disease

RMSF: clinical timing matters

• tick bite (exposure) → 2-14 days later (symptoms begin - fever, headache, GI symptoms) → 3-5 days after fever (rash may appear in hand/soles/trunk)

• the pt may be seriously ill BEFORE detectable antibodies develop

RMSF serology timing problem

• first week

  • pt may have RMSF but antibody may still be undetectable

• 7-10 days

  • antibodies become detectable in many patients

• later

  • serology becomes more useful for confirmation

* BOC TRAP: early negative IFA does NOT rule out RMSF

IFA: serologic gold standard for RMSF

1. R. rickettsii antigen is fixed to a slide

2. add pt serum; specific patient antibody binds to antigen

3. wash, then add fluorescent ant-human immunoglobulin

4. fluorescence under microscope = patient antibody detected

*INDIRECT immunofluorescence → the labeled reagent is the secondary anti-human antibody

paired titers confirm RMSF

• acute specimen

  • early illness

  • example titer - 1:32

• convalescent specimen

  • 2-4 weeks later

  • example titer - 1:128

• 1:32 → 1:128 = FOURFOLD RISE

  • supports recent infection

RMSF older vs current testing

• Weil-Felix

  • detects cross reacting antibody using proteus OX antigens

  • main issue: poor sensitivity/specificity - largely replaced

• IFA

  • detects R. rickettsii-specific IgM/IgG

  • main issue: early specimen may be negative

• Molecular Testing

  • detects rickettsial DNA

  • main issue: direct organism detection - availability/specimen timing matter

knowledge check: a pt has a fever, severe headache, rash, and recent tick exposure. symptoms began 5 days ago. RMSF antibody IFA was negative. what is the best interpretation?

• antibodies may not yet be detectable

  • RMSF antibodies are often undetectable during the first week. timing is the trap

Mycobacterium tuberculosis - what happens after exposure

1. inhalation: aerosolized MTB reaches alveoli

2. macrophage: alveolar macrophages phagocytose MTB. the organism can survive intracellularly

3. cell mediated immunity: T-cell response develops and attempts to contain infection

4. possible outcomes: clearance, latent TB infection (dormant), active TB, later reactivation

TB belongs in immunology bcs control depends heavily on cell mediated immunity

tuberculosis - why T cells matter

• intracellular MTB: macrophage contains MTB antigens

• CD4+ T cell: recognizes presented MTB antigen

• Th1 response: produces IFN-gamma which calls macrophages

• macrophage activation: improved intracellular control of TB

*immune response is measured by PPD (TB skin test) and IGRA (Interferon Gamma Release Assay)

granuloma

• containment, not necessarily elimination

• purpose

  • contain the organism and limit spread - contained does NOT equal eliminated - MTB may persist and can cause latent (dormant) TB infection

• if immune control weakens

  • granuloma containment may fail → MTB replicates → reactivation TB

PPD/TST: measuring cell mediated immunity in vivo

• PPD (pure protein derivative) injected intradermally → previously sensitized T cells recognize antigen → cytokines recruit inflammatory cells → INDURATION develops and is read 48-72 hrs after

• TYPE IV - DELAYED HYPERSENSITIVITY - T CELL MEDIATED

PPD false positive considerations

• BCG vaccinations

• some nontuberculous mycobacteria

  • T cells may recognize related antigens

  • positive TST without MTB infection

PPD false negative considerations

• testing too soon after exposure

• immunosuppression/inadequate immune response

  • patient may be infected but unable to produce detectable induration

*TST detects immune sensitization - not the organism itself

interferon gamma release assay

1. patient blood

   1. bring patient’s blood into the lab

2. MTB antigens

   1. expose sensitized T cells to MTB-specific peptides

3. T-cell response

   1. T cells release IFN-gamma (macrophages engulf and present to T cells)

4. lab readout

   1. measure IFN-gamma response

*IGRA measures a T cell response - NOT MTB DNA and NOT anti-MTB antibody

QuantiFERON-TB

• Nil/NEG control tube

  • baseline IFN-gamma

• TB1 tube

  • MTB antigens → primary CD4 T cell responses

• TB2 tube

  • MTB antigens → CD4 + CD8 T cell response

• Mitogen/POS control tube

  • confirms T cell can respond

• after incubation: collect plasma → measure IFN-gamma by SANDWICH ELISA

  • looking for release of IFN-gamma

why do the QuantiFERON controls matter?

• positive IGRA

  • Nil: low

  • TB antigen: HIGH

  • mitogen: responds

    • T cells specifically responded to MTB antigens

• negative IGRA

  • Nil: low

  • TB antigen: NO response

  • mitogen: STRONG response

    • T cells can respond, but did NOT respond to MTB antigens

• intermediate

  • Nil: low

  • TB antigen: NO response

  • mitogen: NO response

    • cannot tell whether there is no sensitization or the T cells failed to respond

PPD vs IGRA

• both detect immune sensitization - neither directly demonstrates viable MTB

knowledge check: an asymptomatic healthcare worker has a positive QuantiFERON-TB gold plus result. what did the test demonstrate?

• the patient’s T cells recognized MTB antigens and released IFN-gamma

  • a positive IGRA demonstrates immune sensitization. it does not by itself distinguish latent infection from an active disease

final comparison: 4 infections, 4 immunologic testing problems

• GAS vs M. pneumoniae vs RMSF vs TB

• WHAT are we detecting → WHEN was the specimen collected → WHAT does the result actually mean

rapid fire wrap up

• pyoderma/impetigo → Anti-DNase B

• mycoplasma cold agglutinin → IgM anti-I

• early negative RMSF IFA → does NOT rule it out

• positive IGRA → T cell sensitization; NOT proof of active TB

spirochetes big picture

• long, slender, helically coiled bacteria w/ axial filaments (endoflagella)

• characteristic corkscrew motility

• ROUTINE CULTURE IS DIFFICULT

• SEROLOGY therefore plays an important part in diagnosis

  • Treponema pallidum → syphilis

  • Borrelia burgdorferi → lyme disease

  • Leptospira spp. → leptospirosis

  • other Borrelia spp. → relapsing fever

Treponema pallidum

• SYPHILIS

• thin, tightly coiled spirochete

• outer membrane has few exposed proteins and LACKS LPS (usually immunogenic marker)

• cannot be routine cultured in the clinical lab

• transmission: primarily SEXUAL CONTACT; can cross the PLACENTA

4 stages of syphilis

• PRIMARY - painless chancre @ site of inoculation (10-90 days after infection)

• SECONDARY - systemic dissemination; lymphadenopathy, fever, malaise, nonpruritic rash on PALMS/SOLES (hallmark)

• LATENT - no clinical symptoms (early: <1 yr) (later: >1 yr)

• TERTIARY - gummas (big nodules @ site of infection), cardiovascular disease, neurologic complications

neurosyphilis

• do NOT memorize neurosyphilis as a tertiary only complication

• CNS involvement can occur during ANY STAGE

• early manifestations may include meningitis, cranial nerve abnormalities, or stroke

• ocular syphilis and otosyphilis can also occur at ANY STAGE

how can we detect syphilis?

• DIRECT DETECTION

  • detect the organism itself

  • dark field microscopy - direct fluorescent method

• NONTREPONEMAL SEROLOGY

  • detect reagin antibodies associated w/ lipid antigens/tissue damage

  • RPR, VDRL

• TREPONEMAL SEROLOGY

  • detect antibodies against T. pallidum

  • enzyme immunoassays - FTA/ABS, TP-PA, EIA/CILA

direct detection of T. pallidum

• DARK FIELD MICROSCOPY can examine serous fluid from an active lesion

  • collected from chancre

• look for characteristic morphology and corkscrew/flexing motility

• specimen should be examined RAPIDLY

• do NOT use darkfield microscopy on ORAL lesions

  • nonpathogenic oral treponemes may resemble T. pallidum

nontreponemal tests: what is cardiolipin?

• CARDIOLIPIN = phospholipid associated w/ lipid antigens released/exposed during tissue damage and infection

• the immune response produces antibodies historically called REAGIN

  • may include IgM and IgG

• RPR and VDRL detect these anti-lipid/REAGIN antibodies - NOT T. pallidum specific antibody

• the test antigen contains cardiolipin + cholesterol + phosphatidylcholine (lecithin)

RPR and VDRL: flocculation tests

• both are NONTREPONEMAL tests that DETECT REAGIN

• antigen-antibody interaction produces flocculation

• RPR uses CHARCOAL particles so the reaction can be read MACROscopically

• VDRL is read MICROscopically; SERUM requires HEAT INactivation

• VDRL has an important role in CSF testing

  • ONLY METHOD FDA APPROVED FOR NEUROSYPHILIS DIAGNOSIS

• NOT a (+) or (=) report

  • report as: REACTIVE, WEAK REAC., OR NONREACTIVE

why can RPR/VDRL be false positive?

• nontreponemal tests are NOT SPECIFIC for T. pallidum (detects only reagin/damage to cell)

• transient biologic false positives may occur w PREGNANCY, VACCINATION, or INFECTIONS

• chronic false positives may occur w SLE/AUTOIMMUNE disease, IV drug use, ADVANCED AGE, or malignancy

• a reactive nontreponemal test screen must be interpreted w a treponemal test

Prozone false negative

• SECONDARY syphilis may produce very HIGH ANTIBODY concentrations → prozone rxn; sample must be diluted

• antibody excess prevents optimal lattice formation

• little/no visible flocculation → falsely nonreactive result

• solution: perform serial dilutions of the patient’s serum

why report an RPR/VDRL titer?

• reactive samples can be tested in serial twofold dilutions - 1:2 → 1:4 → 1:8 → 1:16 → 1:32…

• titer = highest dilution that remains ACTIVE

• nontreponemal titers usually DECLINE W/ SUCCESSFUL TREATMENT

• a FOURFOLD change is CLINICALLY SIGNIFICANT (successful treatment)

• example - 1:32 → 1:8 = fourfold increase

treponemal tests

• now we get specific

• detect ANTIBODIES directed against T. pallidum ANTIGENS

• examples: FTA-ABS, TP-PA, automated EIA/CILA

• usually become reactive EARLY and remain reactive LONG TERM

  • often remain active AFTER successful TREATMENT

  • therefore: NOT useful for MONITORING RESPONSE to THERAPY

FTA-ABS: indirect fluorescence

• ABS = absorption: patient serum is preabsorbed with NONPATHOGENIC REITER TREPONEMAL ANTIGENS to remove cross-reactive antibodies

• patient antibody binds fixed T. pallidum on the slide

• fluorescent anti-human immunoglobulin binds the patient’s antibody

• fluorescent spirochetes indicate a reactive test

LABEL IS ON ANTI-HUMAN Ig → INDIRECT fluorescence

TP-PA: Particle Agglutination

• colored gelatin particles are coated with TREPONEMAL ANTIGENS

• patient ANTI-T. pallidum ANTIBODIES cross link sensitized particles

• POSITIVE → diffuse lattice/mat (pink spreads throughout well)

• NEGATIVE → compact button (bright pink circle does not spread out in the well)

automated treponemal immunoassays

• high volume labs may use EIA or chemiluminescent immunoassays (CLIA) for TREPONEMAL antibody screening

• automation supports high throughput testing

• treponemal immunoassays are central to the REVERSE screening algorithm

nontreponemal vs treponemal

• NONTREPONEMAL: RPR/ VDRL

  • reagin against cardiolipin-containing antigen → lower specificity → useful for treatment monitoring

• TREPONEMAL: FTA-ABS/ TP-PA/ EIA or CLIA

  • anti-T. pallidum antibody → higher specificity → usually remains active

traditional syphilis algorithm

• SCREEN w/ RPR or VDRL

• if reactive → confirm w TREPONEMAL test

• reactive nontreponemal + reactive treponemal SUPPORTS syphilis

• reactive nontreponemal + nonreactive treponemal suggest a BIOLOGICAL FALSE POSITIVE

reverse syphilis algorithm

• screen w/ AUTOMATED TREPONEMAL EIA/ CLIA

• if reactive → perform quantitative RPR or another NONTREPONEMAL test

• if TREPONEMAL (+) and RPR (=) → perform DIFFERENT TREPONEMAL TEST, commonly TP-PA

*less time consuming, used in large hospital settings

discordant reverse results

• EIA/CLIA (+) and RPR (=) can reflect:

  • previous treated syphilis

  • late/latent infection

  • very early infection

  • false positive initial treponemal screen

• resolve discordance w a different treponemal test such as TP-PA

neurosyphilis CSF testing

• specimen: cerebrospinal fluid

• CSF-VDRL is highly SPECIFC when reactive - only FDA approved test

• a positive CSF-VDRL strongly supports neurosyphilis

• SENSITIVITY IS LIMITED: a negative CSF-VDRL does NOT exclude neurosyphilis

• CSF lymphocytes and protein can provide supportive information

*Positive is Powerful BUT negative does NOT rule it out

syphilis: don’t get tricked

• secondary symptoms + negative RPR → think PROZONE and DILUTE

• positive RPR → confirm w TREPONEMAL testing

• monitor treatment → QUANTITATIVE RPR/ VDRL

• treponemal test (+) AFTER treatment → can be EXPECTED

• EIA (+) / RPR (=) in reverse algorithm → perform TP-PA

• suspected neurosyphilis → CSF-VDRL (negative does NOT exclude)

Borrelia burgdorferi

• LYME DISEASE

• loosely coiled spirochete transmitted by IXODES TICK

• humans = accidental hosts

• nymphs cause many human infections

• transmission generally requires PROLONGED TICK ATTACHMENT

• white footed mouse = important RESERVOIR

• deer = important tick HOST

lyme disease clinical progression

• EARLY LOCALIZED

  • erythema migrans (EM) + flu like symptoms

  • pt may be otherwise symptomatic

• EARLY DISSEMINATED

  • multiple lesions + facial palsy + neurologic involvement + carditis + migratory pain

  • HEMATOGENOUS SPREAD TO MULTIPLE ORGAN SYSTEMS

• LATE

  • ARTHRITIS + PERIPHERAL NEUROPATHY + ENCEPHALOMYELITIS

erythema migrans - why early serology can be negative

• EM may appear before a measurable antibody response develops

• early serology can therefore be nonreactive despite infection

• testing is measuring the HOST antibody response - NOT directly detecting the ORGANISM

• clinical presentation and timing matter

• early localized disease may be present w/ EM while the patient is otherwise asymptomatic

Lyme antibody response

• antibody may not be detectable until several WEEKS AFTER the infection

• IgM appears FIRST; IgG follows and peaks LATER

• early antibiotic treatment may delay or inhibit antibody development

• IgG may persist LONG TERM

  • serology therefore does NOT reliably indicate CURRENT activity or TREATMENT SUCCESS

the 30 day rule for Lyme disease

• symptoms < 30 days: standard immunoblot interpretation may evaluate IgM + IgG

• symptoms > 30 days: rely on IgG

  • isolated IgM is NOT used to support late disease

two tier testing for Lyme disease

• standard two tier testing (STTT)

  • first tier EIA/ IFA → immunoblot if positive/equivocal

• modified two tier testing (MTTT)

  • first EIA → second, different EIA if positive, equivocal

• both approaches use sequential testing to improve interpretation

standard two tier testing (STTT)

• STEP 1: EIA or FIA for B. burgdorferi ANTIBODY

  • negative → usually NO FURTHER TESTING

  • consider TIMING if very EARLY DISEASE

• positive/equivocal step 1 → STEP 2:

  • <30 days → IgM + IgG immunoblot

  • >30 days → IgG immunoblot

what does a Lyme immunoblot band mean?

• Borrelia proteins are represented by molecular size kDa

• patient serum is added to the antigen strip/membrane

• if patient antibody recognizes a particular Borrelia antigen, it binds there

• detection reagent makes that antibody—antigen location visible as a BAND

• the kDa number is the approximate molecular mass of the antigen - NOT the antibody level

lyme immunoblot: reading the pattern

• IgM positive pattern: 2 of 3 designated bands

  • 23 kDa (OspC)

  • 39 kDa

  • 41 kDa (flagellin)

• IgG positive pattern: 5 of 10 designated bands

  • 18, 23, 28, 30, 39, 41, 45, 58, 66, 93 kDa

• DO NOT interpret a single reactive band as diagnostic

why isn’t the 41-kDa band enough?

• 41 kDa = flagellin, an immunodominant antigen

• flagellar proteins can share antigenic similarity among spirochetes

• cross-reactive antibodies may occur w/ other Borrelia species or T. pallidum

• therefore one 41-kDa band alone is not sufficient evidence of Lyme disease