Laboratory Activities 8, 9 and 10 – Non-specific tests for Syphilis: VDRL and RPR Flashcards
is a contagious sexually transmitted disease caused by the spirochete Treponema pallidum subspecies pallidum.
Syphilis
This spirochete causes an immune response in the body by producing antibody-like substance in plasma or serum known as reagin.
Syphilis
[?], a phospholipid derived from beef heart, reacts with reagin, and this antigenic property is used in non-Treponemal tests.
Cardiolipin
NON-TREPONEMAL TESTS AND TRUE-TREPONEMAL TESTS
- Wasserman test
- Direct Detection of Spirochetes
- Serologic Tests
- DNA probe
Direct Detection of Spirochetes
• Darkfield microscopy
• Direct immunefluorescence
Non-Treponemal Serologic Tests
1) Venereal Disease Research Laboratory (VDRL)
2) Rapid Plasma Reagin (RPR)
Treponemal Serologic Tests or True Treponemal Tests
1) Treponema pallidum Immobilization (TPI) test
2) Fluorescent Treponemal Antibody Absorption Test (FTA-ABS)
3) Enzyme immunoassay
4) Hemagglutination Tests
DNA probe
a. Polymerase chain reaction (PCR)
b. Southern blot
SYPHILIS
Caused by:
Treponema pallidum subspecies pallidum (originally called Spirochaeta pallida)
SYPHILIS
Transmission:
Sexual contact, direct blood transfusion or transplacental route
SYPHILIS STAGES
- Primary syphilis
- Secondary syphilis
- Latent syphilis
- Tertiary syphilis
• Associated lesion lasts from 1-6 weeks, during which time the lesion heals spontaneously
- Primary syphilis
• Generalized lymphadenopathy, malaise, fever, pharyngitis, and a rash often involving the mucous membranes and the skin
- Secondary syphilis
• Lack of clinical signs and symptoms
- Latent syphilis
• Manifested by gummatous syphilis, cardiovascular disease, and neurosyphilis
- Tertiary syphilis
o The spirochete is transmitted from mother-to-fetus
Congenital syphilis
Congenital syphilis
o Manifested by the Hutchinsonian triad:
notched incisor teeth, keratitis, and deafness
Congenital syphilis
o Best to use a test that detects IgM antibodies
o Detection of IgM antibodies that are produced by the infant’s immune system
Congenital syphilis
Congenital syphilis
o Treatment:
Arsphenamine or Salvarsan or compound 606 Penicillin
Wasserman test
First diagnostic blood test for syphilis developed in 1906 based on the serologic principle of
o Also known as cardiolipin; identified as a phospholipid (diphosphatidyl glycerol)
Wasserman antigen
o Derived from the host or extract from beef heart
Wasserman antigen
derived from extracts of liver from newborns that had died of congenital syphilis
Wasserman antigen
demonstrated that tissues from animals without syphilis, mainly beef heart extracted in alcohol, could be used equally well as antigens
Landsteiner
• Darkfield microscopy o Positive result:
Corkscrew motility
o Uses anti-treponemal antibody with fluorescent tag against Treponema pallidum antigen in a patient
• Direct immunefluorescence
Non-Treponemal Serologic Tests
o Determine the presence of ____________________
Non-Treponemal Serologic Tests
o Based on the principle of ____________________
Non-Treponemal Serologic Tests
Examples:
• Venereal Disease Research Laboratory (VDRL)
• Rapid Plasma Reagin (RPR)
• Toluidine Red Unheated Serum Test (TRUST)
• Unheated Serum Reagin (USR)
• Reagin Screen Test (RST)
Non-Treponemal Serologic Tests
False-positive: 10-30% may be biologic false positives
infectious mononucleosis, infectious hepatitis, malaria, leprosy, lupus erythematosus, rheumatoid arthritis, advance age, pregnancy
Non-Treponemal Serologic Tests
Positive:
Treponemal infections
Causes yaws: chronic non venereal disease of skin and bones
Treponema pallidum subspecies pertenue
Causes bejel: lesions in the oral cavity, oral mucosa, skin, bones, and nasopharynx
Treponema pallidum subspecies endemicum
Causes pinta: ulcerative skin disease
Treponema carateum
Transmission: traumatized skin comes in contact with an infected lesion
Treponema pallidum subspecies pertenue
Transmission: mouth to mouth by utensils
Treponema pallidum subspecies endemicum
Transmission: traumatized skin comes in contact with an infected lesion
Treponema carateum
Both a qualitative and quantitative slide flocculation test for serum and cerebrospinal fluid (CSF)
Venereal Disease Research Laboratory (VDRL)
Venereal Disease Research Laboratory (VDRL) Antigen consists of:
• 0.03% cardiolipin: main reacting group
• 0.9% cholesterol: enhance the reacting surface of cardiolipin
• 0.21% lecithin: removes anti-complement activity of cardiolipin
main reacting group
• 0.03% cardiolipin:
enhance the reacting surface of cardiolipin
• 0.9% cholesterol:
removes anti-complement activity of cardiolipin
• 0.21% lecithin:
Aside from the VDRL antigen, (?) is also employed
buffered saline containing sodium chloride, formaldehyde, secondary sodium phosphate, and primary potassium phosphate
o Rotation:
• VDRL serum _____ rpm for _____ minutes
• VDRL CSF _____ rpm for _____ minutes
Venereal Disease Research Laboratory (VDRL)
Examination for flocculation:
Microscopic
o 18 gauge needle without bevel that will deliver 60 drops of antigen suspension per ml
Qualitative Serum VDRL
o 19 gauge needle without bevel that will deliver 75 drops of antigen suspension per ml
Quantitative Serum VDRL
o 23 gauge needle that with or without bevel that will deliver 100 drops of saline per mL
Quantitative Serum VDRL
o 21 or 22 gauge needle that will deliver 100 drops per ml
• CSF VDRL
Rapid Plasma Reagin (RPR)
o Reagent: Antigen is similar to the VDRL antigen with the addition of the following:
• Charcoal: allows macroscopic visualization
• EDTA: prevents oxidation
• Thimerosal: preservative
• Choline chloride: inactivates the complement
allows macroscopic visualization
• Charcoal:
prevents oxidation
• EDTA:
preservative
• Thimerosal:
inactivates the complement
• Choline chloride:
RPR
o Specimen
serum
RPR
o Rotation: _____ rpm for _____ minutes
RPR
o Examination for flocculation:
Macroscopic
RPR
o Antigen delivery needle:
• 20 gauge disposable needle without bevel, 60 drops are obtained in 1 ml
Detect Treponemal antibodies
Treponemal Serologic Tests or True Treponemal Tests
Involves mixing of patient serum with live, actively motile Treponema pallidum extracted from testicular chancre of a rabbit
Treponema pallidum Immobilization (TPI) test
o Patient serum is heat-inactivated and made with a sorbent consisting of nonpathogenic treponemes (Reiter strain) which removes the cross-reactivity with treponemes other than Treponema pallidum
Fluorescent Treponemal Antibody Absorption Test (FTA-ABS)
o Nichols strain of Treponema pallidum has been fixed to slides used for the test
Fluorescent Treponemal Antibody Absorption Test (FTA-ABS)
For detection of anti-treponemal antibody from a patient
Enzyme immunoassay
Antigen: RBCs sensitized with Nichols strains
Hemagglutination Tests
Hemagglutination Tests
• Hemagglutination Treponemal Test for Syphilis (HATTS)
• Treponema pallidum Hemagglutination Assay (TPHA)
• Microhemagglutination Assay for Antibodies to Treponema pallidum (MHA-TP)
o Reagent: Sheep RBCs coated with antigens from Nichols strain of Treponema pallidum; Sorbent
Hemagglutination Tests
Hemagglutination Tests
o Positive reaction:
Rough or jagged pattern of cells in microtiter wells
• Patient DNA matched with treponemal DNA
DNA probe
• A means of amplifying tiny quantities of nucleic acid using a heat-stable polymerase enzyme and a primer that is specific for the DNA sequence desired
Polymerase chain reaction (PCR)
Technique for the identification of specific DNA sequences in which DNA is cleaved into fragments by enzymes, separated electrophoretically, denatured, transferred to a nitrocellulose membrane, and incubated with a labeled probe that is specific for the sequence of interest
Southern blot
is a protein found in blood plasma that shows increased levels in response to inflammation.
C-reactive protein (CRP)
It is synthesized in the liver and has a half-life of 20 to 30 hours.
CRP
It belongs to a group of proteins known as acute phase reactants whose concentrations increase during a general, nonspecific response to infectious and inflammatory processes, cellular necrosis, and malignant neoplasia.
CRP
contributes to non-specific immunity by activating complement and stimulating phagocytosis.
CRP
A nonspecific, acute-phase, reactant glycoprotein that increases very significantly with the onset of a substantial inflammatory stimulus, such as infection, myocardial infarction, or surgical procedures
C-reactive protein (CRP)
CRP Common serologic test:
Latex agglutination
(?) are reacted with patient serum. In this case, the CRP is acting as the antigen. If CRP is present above normal threshold levels, the antigen antibody combination will result in a (?)
Latex particles coated with antibody to CRP
visible agglutination reaction
sensitive, although nonspecific, indicator of inflammation
elevated CRP level
Normal levels range from __________ in newborns to __________ in adults
CRP
gives a positive result with the undiluted specimen
0.6 mg/dL or higher
CRP levels can also be used to monitor the outcome of
surgery, graft rejection, drug therapy with anti-inflammatory agents, and recurrence of malignancies
is a chronic disease that affects the joints characterized by inflammation, stiffness, and soreness.
Rheumatoid arthritis (RA)
Most individuals affected by (?) experience a gradual onset of the disease, but usually, all joints in the body will be affected.
rheumatoid arthritis
As Rheumatoid arthritis progresses, other organs of the body can develop an
inflammatory condition.
The determination of the rheumatoid factor is essential in the (?) of rheumatoid arthritis
prognosis and therapeutic management
• An anti-antibody that binds to the Fc portion of abnormal IgG
• Associated to rheumatoid arthritis
Rheumatoid factor (RF)
o Chronic systemic inflammatory disorder in which joint cartilage, ligaments, and tendons are destroyed
rheumatoid arthritis
o An autoimmune disease that affects the synovial membrane of multiple joints
rheumatoid arthritis
Also noted in chronic hepatitis, syphilis, systemic lupus erythematosus (SLE), scleroderma, Sjogren’s syndrome, and Bcell lymphoproliferative disorders
Rheumatoid factor (RF)
• Principle:
• Passive agglutination can be employed to test for the presence of the antibody, using a carrier particle such as (?) with IgG. When (?) is mixed with the sensitized reagent cells, visible agglutination occurs.
sheep erythrocytes sensitized
patient serum containing RF
Latex fixation test by
Singer and Plotz
Latex agglutination titer lower limit based on an undiluted sample and diluted sample at
1:10
• Very low titer (present also in other diseases such as SLE, endocarditis, tuberculosis, syphilis, viral infection, and diseases of the liver, lung, or kidney)
o If positive only in undiluted
• Low titer can be found in approximately 1 percent of healthy individuals
o If positive only in undiluted
• RF is present in the specimen at a level generally associated with RA
o If positive at 1:10 dilution
• Interpretation of results based on titer:
o Positive: ____________________
o Weakly positive: ____________________
o Negative: ____________________
Rheumatoid factor (RF) Methods
- Latex agglutination
- Sheep cell agglutination test: Rose-Waaler Test
- Sensitized alligator erythrocyte test: Cohen et.al
- Bentonite flocculation test: Bloch and Bunim
is useful in the diagnosis of Streptococcal infection and its relative effects on organs, especially the heart and kidneys.
Anti-Streptolysin O (ASO) test
Though the causative agent of Group A Streptococcal infection can be cultured, it can also be detected through serologic techniques.
Anti-Streptolysin O (ASO) test
is a gram-positive coccus responsible for several human infections, some of which can have serious sequelae
Streptococcus pyogenes
: Major virulence factor for Streptococcus pyogenes; more than 60 M serotypes have been identified
o M protein
• Virtually all strains of Streptococcus pyogenes produce two hemolysins
o Bacterial toxins
• Enzyme that causes hemolysis by binding to cholesterol in the RBC membrane
- Streptolysin O (SLO)
• Enzyme that causes hemolysis by disrupting the selective permeability of the RBC membrane
- Streptolysin S
caused by Streptococcus pyogenes include cellulitis, impetigo, and erysipelas
Skin infections
caused by Streptococcus pyogenes are characterized by fever, sore throat, and pharyngeal edema
Upper respiratory tract infections (URTI)
caused by a strain of Streptococcus pyogenes that produces an erythrogenic toxin, which results in a characteristic rash; fever and sore throat are also present
Scarlet fever
a complication seen following upper respiratory tract infections
Rheumatic fever (RF)
o All M serotypes that cause pharyngitis have been implicated in
RF
o results in damage to heart valves, and patients with rheumatic heart disease have an increased risk of developing endocarditis and other cardiac problems in later years
RF
may occur after pharyngitis or skin infections
Poststreptococcal glomerulonephritis
o Only a few M serotypes cause this type of glomerulonephritis; patients have an increased risk of developing renal failure later
Poststreptococcal glomerulonephritis
• The mechanisms by which these (?) occur are not fully understood
sequelae
• Antibodies to streptococcal cell membranes cross-react with (?) in cardiac muscle cells, which results in cell damage
myosin
• Antigen-antibody complexes form at the glomerular basement membrane and attract inflammatory cells that cause
renal tissue damage
During infection with Streptococcus pyogenes, SLO produced by Streptococcus pyogenes elicits an immune response, and specific antibodies are formed→ these antibodies neutralize the
hemolytic activity of the SLO
The ASO titer begins to increase approximately (?) and peaks (?)
7 days after infection
after 4 to 6 weeks
SLO is added to serial dilutions of patient serum, along with (?) as indicator cells → if the patient serum contains antibodies against SLO, the antibodies will complex with the corresponding antigens → these complexes block the hemolytic activity of the antigen, and no hemolysis occurs
group O RBCs
• The ASO titer is reported as the reciprocal of the highest dilution that shows no hemolysis and is expressed in
Todd units
o Normal: titer of __________ Todd units
o Moderately elevated: titer at least (?) in an adult and (?) in a child
240 Todd units
320 Todd units
• High titers with Streptococcal infections, rheumatic fever, glomerulonephritis
Anti-streptolysin O (ASO) titer
Based on the neutralization of the hemolytic activity of streptolysin O
Conventional method:
Conventional method: Positive Resul
Serum with Ab (ASO) → + streptolysin O reagent → neutralization of streptolysin O by patient Ab (ASO) → + RBC (indicator) → _________________________ (presence or absence of hemolysis?
Conventional method:
Negative Result
Serum without Ab (no ASO) → + streptolysin O reagent ® neutralization of streptolysin O by patient Ab (ASO) → + RBC (indicator) → _________________________ (presence or absence of hemolysis?)
Conventional method Controls:
RBC control (RBCs + buffer) should show NO hemolysis
Streptolysin control (streptolysin O + buffer + RBCs) should show hemolysis
Rheumatoid factor (RF)
Common methods:
Nephelometry
Latex agglutination
sometimes appear earlier than Anti-streptolysin O in streptococcal pharyngitis
Anti-DNase B
are increased in the 15% to 20% of RF patients who do not have elevated ASO titers
Anti-DNase B levels
Sensitivity is increased for the detection of glomerulonephritis preceded by streptococcal skin infections as this type of disease does not stimulate ASO antibodies
Anti-DNase B testing
Measurement is based on neutralization methodology
Anti-DNase B testing
If anti-DNase B antibodies are present, they will neutralize reagent DNase B, preventing it from depolymerizing DNA → presence of DNase is measured by its effect on
DNA-methyl green conjugate
Slide agglutination screening test for detection of antibodies to several streptococcal antigens
Streptozyme testing
Principle: Sheep RBCs are coated with antigens so that antibodies to any of the streptococcal antigens can be detected
Streptozyme testing
Several rapid tests are now available to detect streptococcal antigens
The advantage is their (?) over other test methods; however, a significant number of false-negative results occur
speed
