Serology cont'd

Legionnaire's disease, febrile agglutinins, S. aureus, GAS, M. pneumoniae, Spirochetes

Legionella: m/c are serogroup 1, 3, 4, and 6, which is most pathogenic?

serogrp 1

Legionella char

slender, pleo, GNR, faint staining in GST

req L-cysteine & iron (BCYE w/PAC or PVA): looks ground glass

Legionella epidemiology

bodies of water, air conditioning ducts, can resist disinfectants, can parasitize amoebae in water

L. pnuemophila pathogeneisis

Legionellosis: fever, chills, dry nonproductive cough → affects GI, CNS, liver, kidneys

Pontiac fever: self limiting febrile illness, hypersensitivity rxn to organism

Legionella lab ID

culture

direct Ag: DFA, urine Ag

serology: indirect fluor Ab

Legionella: direct Ag DFA

rapid but less sensitive than cx

1) pt specimen + monoclonal

2) incub 30min at 37C

3) add mounting media

4) read under fluorescent microscope

Legionella: urine Ag

EIA method, 15min

Ab+Ag for L. pneumophila serogroup 1= positive line

Legionnaire’s dz → shed Ag in urine

may be false neg early in dz

Legionella: serology IFA

sandwich assay

1) Ag (L.pneumophila) + diluted pt serum (Ab) → incubate, rinse

2) Ag-Ab complex + anti-human globulin w fluorescent tag → incub, rinse → read

results: highest dilution of pt serum showing 1+ fluorescence = pts positive titer

sample: acute serum should be obtained w/in 1 week pre or post onset & convalescent from 3 to 6 weeks after onset.

other lab tests for L. pneumophila

molecular assay: PCR or nucleic acid test are available but v $$, usually confirmatory

immunological methods: latex agglutination allows rapid elimination of NEG samples, POS can be checked out w more targeted tests

also not FDA-approved

Legionella lab ID adv vs disadv

febrile agglutinin

agglutinating-Ab that arise from infns from orgs that induce FEVER

often used to dx dz where bug is difficult to grow in vitro

ex) Francisella (tularemia),

Rickettsia (typhus),

Brucella (brucellosis),

Salmonella (typhoid fever)

febrile Agglutinin test (FAT): Widal test

detects Ab to typhoid & paratyphoid fever caused by Salmonella spp.

FAT: Weil Felix test

based on x-reaction of Ricketssia -Ag w O-Ag polysaccharide in Proteus vulgaris strains OX2/19/K

→ measures anti-rickettisial Ab in pt serum by agglutination w/Proteus bacteria

**cannot distinguish b/t R. prowazekii, typhi, rickettsii

FAT: slide agglutination test

test Ab in pt’s serum

steps: 80uL test sample + 1 drop of Febrile Ag (40 uL) → mix & rotate → read after 30 sec

no agglutination = NEG → titer is <1:20

agglutination 2+ or greater = POS → set up tube agglutination for confirmation

FAT: tube agglutination test

steps: serial dilutions of POS pt’s serum in saline, 1:10 → 1:2560 → add 0.5mL Ag & incubate in water bath

tube agglutination results

• what are the 2 types of agglutination rxns?

read degree of clarity in supernatant

gently tap tube to suspend agglutinated particles

somatic agglutination: usually granular & will settle quickly to bottom of tube

flagellar agglutination: more floccular , fluffy agglutinate that are readily dispersed

tube agglutination interpretation

Ab due to infn may not be detected early in infn

max titer levels occur usually at convalescence

neg test are NOT conclusive

pos result may be due to vaccination

most conclusive serological evidence of infn = at least 4-fold inc in serum drawn b/t acute & convalescent phase of illness or a single titer >1:80 to 1:160

febrile agglutinins table

after infn w/S.aureus → body produces Ab to key cell wall component called __.

teichoic acid

Teichoic acid assay: method

Ouchterlony double diffusion technique

Ag & Ab diffuse in medium

→ bands forming = Ab-Ag complex formation, density of lines indicate amount of complex

teichoic acid assay titers

1:2 seen in normal individuals, transient S. aureus infns, pts w other G+ infns

1:4 or greater have been seen in: EC pts, deep seated infns, non-EC pts

titer = highest titer w/ band of ID

Strep pyogenes: pathogenesis

m/c cause of bacti pharyngitis

after inital infn → Ab made x-react w skeletal, smooth muscle, and myocardial fibers causing 2 sequelaes: rheumatic fever & glomerulonephritis

What is S. pyogenes main virulence factor?

M-protein = limits phagocytosis

sequelae to GAS infn: rheumatic fever

assoc’d w GAS pharyngitis (NOT w skin infns)

usually in kids 6-15 y/o, occurs 20 days after scarlet fever or strep throat

symptoms include joint pain, fever, heart problems, shortness of breath → permanent heart damage

serological findings are assoc’d w elevated ASO, anti-DNase, and/or anti-hyaluronidase

acute glomerulonephritis

assoc’d w respiratory AND skin infns → 10 days after GAS infn

caused by deposition of Ag-Ab complexes in glomerular capillary walls → edema, hypertension, hematuria, proteinuria

GAS lab ID relies on 3 Ab produced:

1) streptolysin O (ASO)

2) deoxyribonuclease B (anti-DNase B)

3) anti-hyaluronidase (anti-AHT)

anti-streptolysin O (ASO)

O2-labile enzyme from most GAS throat/respiratory infns; is a hemolysin that lyses rbc, wbc, plt, cultured cells

b/c ASO is irreversibly inhibited by cholesterol in skin lipids, pts w GAS skin infn do NOT develop anti-ASO Ab

other GAS extracellular enzymes formed

Streptolysin S = O2-stable, non-antigenic (no Ab)

Strepokinase = lyses formed clots

anti-Streptolysin O (ASO) test

neutralization tube test: v time consuming

latex agglutn test: rapid, false pos/neg are common

nephelometry: measures light scattered from Ab-Ag

deoxyribonuclease B (anti-DNase B)

DNase enzymes that degrades DNA, not cytolytic → reduced viscosity of abscess material → facilitates spread of bug

** marker for GAS skin infns since fail to make anti-ASO

Ab remains elevated for months & dec slowly

deoxyribonuclease B assay

titers inc after skin infns

test measures Ab ability to neutralize DNase → which has a end point color indicator

POS = blue (no change, enzyme neutralized by Ab)

NEG = pink = DNA is depolymerized

can make serological titers (quantitative)

anti-hyaluronidase (AHT) (aka “spreading factor'“)

inc after GAS throat & skin infns

depolymerize hyaluronic acid (constituent of synovial fld & other connective tissue), one produced by GAS is immunologically distinct (is heat-stable & doesn’t req Mg2+)

POS result = important for rheumatic fever dx

AHT assay

when hyaluronidase is present → will hydrolyze hyaluronic acid if no Ab to neutralize → no mucin clot

POS for Ab = mucin clot

NEG = no clot

titer = reciprocal of highest pt dilution showing a clot

better indicator of rheumatic fever than ASO test

pt w rheumatic fever may have titer of 1:1024 and greater

titer = 1:16

Mycoplasma pneumoniae char

smallest free-living bacteria, doesn’t GST

no cell wall → penicillin-R

v fastidious to grow

“fried egg” colony morph

→ causes “walking pneumonia”

Mycoplasma pneumoniae serological ID

standard is complement fixation (CF) → poor sensitivity

cold agglutinin assay shows 65% sensitivity (but poor specificity)

cold agglutinins for M. pneumo

cold autoimmune IgM Ab that bind at 2-4C → cause rbc to clump tg when person is exposed to cold temps → inc chance affected rbc will be destroyed by the body

bind to blood group I/i-Ag found in rbcs (auto anti-I can be stimulated by org that carry an I-like Ag on their surface, ie M. pneumo or anti-i in infectious mono)

cold agglutinins not specific bc

x-reactivity w EBV (mono), CMV, adenovirus, other disease lymphoma

strong reactive cold agglutinin titer of 1:128 or significant titer increase = recent infn w M. pneumo

Syphilis mode of transmission

cannot grow in vitro

spread via direct contact (via sex) 30-50% chance if partner has active lesion

congenital infns also

primary syphilis

lesion = chancre, painless

usually outside penis, vagina or cervix

secondary syphilis

40% pt have neurologic signs ie visual disturbances, hearing loss, tinnitius facial weakness

stage where org is most numerous

all sero tests = POS

latent (hidden) syphilis

after 12 mo

pt are non-infectious (except pregnant women)ter

tertiary syphilis

gummatous syphilis

cardiovascular

neurosyphilis

gummas

localized areas of granulomatous inflammation

cardiovascular syphilis

ascending aorta → aortic aneurysm

neurosyphilis

m/c complication of tertiary syphilis, acute MG → tabes dorsalis

large rise in HIV+ pt

congenital syphilis

fetal death 10%

60-90% develop cluttons joints, deafness, Hutchinson’s teeth, mulberry molars & bone abnormalities

EIA test for syphilis

used for large vol testing → lowers manual labor; detects Ab to lipoproteins from damaged cells & cardiolipin from treponemes & thus are NOT specific for Treponema (Liason IgG & IgM)

is it non-trep OR trepeneme specific?

syphilis EIA results

if EIA POS → RPR (rapid plasma reagin)

if NEG → do TPPA (confirmatory test)

if RPR POS → titer RPR via serial dilutions of serum (but can have false POS due to infectious mono, lupus, pregancy)

nontreponemal sero tests: RPR

CANNOT perform on CSF

Ag = cardiolipin (w choline chloride), cholesterol, lecithin, charcoal added for macroscopic viewing

nontreponemal sero tests: VDRL

venereal disease research laboratory

performed on CSF (or serum) → use for neurosyphilis dx

Ag = cardiolipin, cholesterol, lecithin

microscopic flocculation 100x

nontreponemal sero tests

Ab appear 1-4 weeks after primary chancre, usually highest in titer during secondary syphilis, titer gradually dec after 1st year

used to screen/monitor course of dz, efficacy of treatment

4-fold change in titer = change of 2 dilutions → considered necessary to demonstrate a clinically significant difference

• from 1:16 to 1:4 or from 1:8 to 1:32

reagin

an antibody-like substance present in serum from pt w/syphilis

binds to Ag cardiolipin-lecithin coated particles → flocculation

neurosyphilis dx

need POS serum treponemal test & POS VDRL in CSF

CSF VDRL is specific, but not sensitive

treponemal test

detect Ab directed against T.pallidum organism

more difficult to perform, used for confirmation

types
- fluorescent treponemal Ab absorbed (FTA-ABS)

-agglutination test

darkfield microscopy for syphilis dx

organism present in lesion & exudates, false NEG if transport to lab is delayed (dies easily)

pathogenic treponemes are ID’d based on corkscrew motility & flexing motility = live specimen req’d

fluorescent Ab for syphilis dx

utilizes fluorescent-labeled Ab

sensitive & specific compared to darkfield

live specimen NOT req’d

but x-reactivity w related treponemes

fluorescent treponemal Ab absorbed (FTA-ABS)

slides have Nichols strain of T.pallidum

uses Reiter Treponemes to remove reactivity w lupus Ab → prevents x-reactivity w Ab to other treponemes

fluro 2+ or greater = POS

highly specific for treponemal Ab, 100% reactive in secondary/latent syphilis

agglutination treponemal test: TPPA

Treponema pallidum particle agglutination

performed if RPR=NEG for confirmation

pt serum is incubated w either sensitized T. pallidum gel particles or unsensitized gel particles as a control

POS= smooth mat that covers well’s surface due to formation of lattice-like structure

NEG = form compact button

Lyme disease caused by

Borrelia burgdorferi

loosely-coiled spirochete

can be cx w Barbour-Stoenner-Kelly media

vector = Ixodes tick

arthritis → skin, heart, joint involvment

Lyme disease stages

1) localized rash: erythema migrans, bullseye rash, fades untreated

2) early dissemination: in blood, skin, CNS, heart, joints afected

3) late dissemination: arthritis, late neuroborreliosis

Lyme dz dx: immunofluorescence

detects IgM or IgG, v subjective

Lyme dz dx: EIA

rapid, specific, insensitive in early stage of dz

Lyme dz dx: Western blot

confirmation test after POS EIA/IFA

Lyme dz dx: PCR

highly specific, but specificity varies by source; most sensitive in synovial fluid

least sensitive in CSF