BACTERIAL INFECTIONS - SEROLOGICAL TESTS FOR SYPHILIS Flashcards
- Microscopic due to absence of visualizing agent
VDRL
- Both a qualitative and quantitative slide flocculation test for serum and spinal fluid (neurosyphilis)
VDRL
Antigen (reagent) consists:
- CARDIOLIPIN (0.03%)
- Lecithin (0.21%)
- Cholesterol (0.9%)
Serves as Ag
- CARDIOLIPIN (0.03%)
Main reacting component
- CARDIOLIPIN (0.03%)
Neutralize the anti-complementary properties of cardiolipin
- Lecithin (0.21%)
Increase the reactive surface and complement fixing capacity of cardiolipin
- Cholesterol (0.9%)
- Unknown is reagin Ab, w/c specifically reacts w/ cardiolipin
VDRL
Inactivated serum: [?]
56°C heat for 30 mins
to destroy the complement
Inactivated serum
Volume of the sample:
50 ul
Serum:
14mm slide with ceramic ring
o 180 rpm for 4 mins
Spinal fluid:
16 mm in diameter and 1.75mm depth
o 180 rpm for 8 mins
- Qualitative Serum VDRL:
18-gauge needle without bevel that will deliver 60 drops of antigen suspension per mL; Ring: 14mm
- Quantitative Serum VDRL:
19-gauge needle without bevel that will deliver 75 drops of antigen suspension per mL; 23-gauge needle that with or without bevel will deliver 100 drops of saline per mL; Ring: 14mm
- CSF VDRL:
21- or 22-gauge needle that will deliver 100 drops per mL; Ring: 16 mm
Macroscopic due to presence of charcoal as visualizing agent
RPR
Unheated serum
RPR
Standard test for screening Syphilis
RPR
RPR Principle:
RPR Ag + 1egain (if present) will combine with lipids and coagglutinates with charcoal
RPR Endpoint:
Flocculation (black clumps)
Modified VDRL Ag - reagent
RPR
- Original VDRL Ag (?)
CARDIOLIPIN
Main reacting component in RPR
CARDIOLIPIN
: enhances the ability of the suspension by inhibiting the oxidation of lipids
- Na2EDTA
: visualizing agt
- CHARCOAL
: buffers the suspension
- PHOSPHATE
: preservative
- THIMEROSAL
also inactivates the complement
- THIMEROSAL
: eliminates the need for heating because it inactivates the inhibitors in serum or plasma
- CHOLINE CHLORIDE
Primarily inactivates the complement
- CHOLINE CHLORIDE
FTA-ABS Principle:
Indirect IF test
FTA-ABS Unknown:
Ab
FTA-ABS Indicator label:
FITC
Heat inactivated patient serum reacts with Sorbent consisting of Nonpathogenic treponemes (?)
Reiter strain
to remove cross-reactivity
Reiter strain
Perform indirect IF using pathogenic strain (?) fixed to slide
Nichols strain
FTA-ABS (+) result
(+) Green fluorescence = Presence of Anti-T. pallidum Ab
T. pallidum subspecies pallidum causes:
Syphilis
Bejel
Pinta
Use [?] to remove crossreactivity to other diseases and confirm syphilis
reiter strain
If Anti-T. pallidum Ab is present, it will react with/ [?]
Nichols strain
HEMAGGLUTINATION TESTS Principle:
Agglutination
RBCs are sensitized with Nichols strain
HEMAGGLUTINATION TESTS
HEMAGGLUTINATION TESTS Reactive result:
sooth matte at the bottom of the reaction well
HEMAGGLUTINATION TESTS Nonreactive result:
compact button of cells with or without a hole in the center
Only performed by reference lab requiring expertise
HEMAGGLUTINATION TESTS
- ТРНА:
Turkey erythrocytes
- МНА-ТР:
Tanned sheep erythrocytes
Lyme disease Clinical hallmark:
(chronicum/classic) Erythema migrans (EM)
Erythema migrans (EM) characteristics
Bull’s eye rash
Tick burrowing
Lyme disease bacterial agent
Borrelia burgdorferi
Lyme disease Vector:
Ixodes scapularis tick – reduviid bug
MOT: vector-borne
Lyme disease
Survives in hot temperature
Ixodes scapularis tick – reduviid bug
Resembles signs and symptoms of stroke (facial palsy), gouty arthritis (swollen knee)
Lyme disease
Febrile disease – extremely high fever (what temp range?)
> 37.5oC or 39 to 40oC
Group A Streptococcus agent
Streptococcus pyogenes
Streptococcus pyogenes causes:
- URTI –pharyngitis: main infection
- Necrotizing fasciitis: flesh-eating bacteria
- Streptococcal Toxic Shock Syndrome
- Streptococcal pyoderma (impetigo)
- Scarlet fever
- Erysipelas
(following infection after main infection)
sequelae
Most important sequelae
- Acute Rheumatic fever
- Post streptococcal Glomerulonephritis
Acute Rheumatic fever
develops as a sequela to pharyngitis or tonsillitis in [?] of infected individuals.
2 to 3 percent
Acute Rheumatic fever
period is typically [?] after onset of the sore throat.
1 to 3 weeks
Characteristic features of acute rheumatic fever include
fever inflammation in the joints, and inflammation of the heart
Acute Rheumatic fever Virulent factor:
M protein
attaches itself to heart valves; forms Ab against M protein where it attaches leading to Ag-Ab reaction; attacks the heart
M protein
• condition characterized by damage to the glomeruli in the kidney due to the deposition of Ab-streptococcal immune complexes
Post streptococcal Glomerulonephritis
Kidneys: filters the blood
Glomerulus - first filtering part
Ag-Ab does not pass thru and accumulates in the glomerulus
Result: Nephritis
Post streptococcal Glomerulonephritis
Immunologic Features
- Lipoteichoic Acid
- Protein F
- M Proteins
: adheres to epithelial cells ‘
- Lipoteichoic Acid
: adheres to epithelial cells
- Protein F
: anti-phagocytic, mimic proteins on the heart valve
- M Proteins
(Virulence Factors: substances that can produce Abs against)
Immunologic Features
Cannot be easily recognized by the immune system
M Proteins
Mimics the protein on the heart valve
M Proteins
Extracellular products - aka
exoantigens
Extracellular products
• Hemolysins (Streptolysin O and S)
• Hyaluronidase
• DNAse
• Streptokinase
• Erythrogenic toxin
- spreading factor
Hyaluronidase
– exoantigen tested in the sero lab
DNAse
causes scarlet fever
Erythrogenic toxin
Strawberry tongue
Erythrogenic toxin
Ag or Ab
- Reagent
ANTIGEN
Ag or Ab
- Unknown in the px
ANTIBODY
Whole or processed B. burgdorferi
IFA
Anti-Borrelia antibody from patient, antihuman globulin with fluorescent tag
IFA
Difficult to perform
IFA
Sonicated B. burgdorferi
EIA
Anti-Borrelia antibody from patient, antihuman globulin with enzyme tag
EIA
Easy to perform
EIA
Purified flagellin protein
EIA
Antiflagellin antibody from patient, antihuman globulin with enzyme tag
EIA
Easy to perform; highly specific; sensitive in early Lyme disease
EIA
C6 peptide
EIA
Conserved region of surface lipoprotein (VlsE)
EIA
Easy to perform; highly specific; initial studies promising; sensitive in early and late Lyme disease
EIA
Patient DNA matched to Borrelia DNA
DNA probe
Ag: None
DNA probe
Technically demanding, very specific; lacks sensitivity
ANTISTREPTOLYSIN O TEST (ASTO/ASO) Principle:
based on the neutralization of the hemolytic activity of streptolysin O
ANTISTREPTOLYSIN O TEST (ASTO/ASO) Principle in the lab:
Latex Agglutination
If a patient’s serum containing antistreptolysin O Ab is added to the streptolysin Rgt, an Ag-Ab reaction occurs causing the neutralization of the toxin
ANTISTREPTOLYSIN O TEST (ASTO/ASO)
ANTISTREPTOLYSIN O TEST (ASTO/ASO)
Positive result:
Negative result:
Positive result: NO LYSIS
Negative result: Hemolysis
Patient serum: w/ anti-SLO Ab
Reagent:
1. SLO
-SLO + anti-SLO = Ag-Ab binding
-SLO rgt will be neutralized
2. RBC (indicator)
-Di papansinin
-(+) Result: No lysis due to presence of anti-SLO Ab
What is the result?
Positive result: NO LYSIS
Patient serum: w/o anti-SLO Ab
Reagent:
3. SLO
-No neutralization
-SLO rgt will be hydrolyzed
4. RBC (indicator)
-SLO + RBC = Lyse
-(+) Result: Lysis due to absence of anti-SLO Ab
What is the result?
Negative result: Hemolysis
– caused by toxin or SLO
o Dirty purple
– caused by complement
o “vin rose” / red lysis
What tube is (+) control in ASO?
Tube 13
What tube is (-) control in ASO?
Tube 14
is the RBC control (no hemolysis)
Tube 13
is the SLO control (hemolysis)
Tube 14
ASO
Prepare [?] tubes
14
ASO Serum Dilution
0.5ml serum + 4.5ml of diluent
ASO Normal:
Titer of 166 Todd units or below (0-166)
ASO Moderately elevated
-Adult:
-Child:
240 Todd units
320 Todd units
o Passive agglutination
ASO Latex Agglutination Slide Method
ASO Latex Agglutination Slide Method NV:
<200IU/mL
ASO Latex Agglutination Slide Method
(+) Result:
Agglutination
ASO Latex Agglutination Slide Method False (+) on px w/
braces
ANTI-DNASE B TESTING Principle:
Neutralization
ANTI-DNASE B TESTING
If [?] is present, it will neutralize the rgt [?]
Anti-DNAse B Ab
DNASE B
ANTI-DNASE B TESTING Positive result:
green complex (Intact DNAmethyl green conjugate because no hydrolysis)
ANTI-DNASE B TESTING Negative result:
hydrolysis of the green conjugate to colorless
Reagents:
1. DNASE B
2. DNASE B rgt will not be neutralized due to absence of Ab DNAmethyl green
- DNASE B rgt has now the capability to hydrolyze the DNAmethyl green resulting to a colorless cpd
-(-) Result: due to absence of Anti-DNAse B
What is the result?
Positive result: green complex (Intact DNAmethyl green conjugate because no hydrolysis)
Reagents:
1. DNASE B = Ag-Ab binding
- DNASE B rgt will be neutralized
2. DNAmethyl green
- Cannot be hydrolysed since it is bound to Ab
What is the result?
Negative result: hydrolysis of the green conjugate to colorless
slide agglutination screening test
STREPTOZYME TESTING
detection of antibodies to several streptococcal antigens
STREPTOZYME TESTING
STREPTOZYME TESTING
Sheep red blood cells are coated with
streptolysin, streptokinase, hyaluronidase, DNase, and NADase
STREPTOZYME TESTING
Positive test:
Hemagglutination
false positives
IFA
EIA
subjective
IFA
more sensitive than IFA
EIA
