Oh my god. How time flies. This is the last 5 weeks of our SIP already. Before we know it, we’ll be back in school tackling tutorial and lecture quizzes and term tests. Ok then, let me cut the chase and get on with my post.
Anyway, for the past 2 weeks, I have been attached to the Serology department. Within this department, there are 2 labs: STD lab and Serology lab. In the STD lab, tests for syphilis and gonorrhoeae are done. But I think the gonorrhoeae section will be moved to the Bacteriology department at the beginning of November. In the serology lab, tests for autoimmune and infectious disease are done. But for this post, I shall just concentrate on the tests for syphilis.
Let me give a brief introduction to syphilis. I am sure you all know that syphilis is a sexually transmitted disease. It is caused by bacteria known as Treponema pallidum. This organism belongs to the order Spirochaetales and is a member of the Treponemataceae family. It is a thin, delicate organism with tapering ends. Its size can vary from 6 to 15 microns in length with a thickness of around 0.25 microns. The organism also has 6 to 14 spirals with pointed ends that have finely spiral terminal filaments. Syphilis can be divided into several stages: early syphilis, late syphilis and congenital syphilis. Early syphilis is the initial stage of infection while late syphilis occurs only 10 to 20 years after initial infections. Congenital syphilis is not connected to early or late syphilis. It is syphilis infection in infants.
Tests for syphilis can be divided into non-treponemal and treponemal tests.
Non-treponemal test
Non-treponemal tests are screening tests as they screen patients for non-specific reagin antibodies that are produced during an infection. The antigens used for screen are lecithin and cardiolipin. When treponemas is present, the lipid from the cell surfaces would cause the host to produce anti-lipid IgG and IgM antibodies. So the lecithin and cardiolipin antigens will bind to these antibodies, forming complexes, which will stay suspended in the tests via flocculation. The type of non-treponemal test used in the lab is the Venereal disease research laboratory (VDRL) test.
Venereal disease research laboratory (VDRL) test
Principle:
VDRL test is a slide microflocculation test for syphilis. When treponemas is present in the host, anti-lipid IgG and IgM antibodies known as reagin will be produced, in response to the lipid on the treponemal surface. These antibodies will then react with substances on the mitochondrial membrane. The antigen used in this test is mixed with buffered saline and consists of cardiolipin, cholesterol and lecithin. These substances will react with the reagin present in the serum of syphilitic patient.
A reactive test means that flocculation, a combination of principles of precipitation and agglutination, has occurred when the antigen reacts with antibody, thus forming antigen-antibody complexes. These complexes can be viewed as big clumps under the microscope. A weakly reactive test means that the clumps are smaller than that of a reactive test. A non-reactive test means that no clumps are observed under the microscope.
The test is divided into qualitative test and quantitative test. For qualitative test, it just indicates whether the sample is reactive or not. For the quantitative test, dilution of the serum will be done. So the highest dilution with a reactive result can be known.
However, as reagins may be produced in other treponemal diseases and in response to a non-treponemal disease, which results in tissue damage, a reactive VDRL test does not confirm a T. pallidum infection resulting in syphilis. But for a reactive VDRL test, a treponemal test (e.g TPPA) would follow to confirm the reactive result.
Procedure:
Qualitative test
1. Spin the blood at 3000 rpm for 10 minutes.
2. Transfer 50 uL of serum onto 1 ring of the ceramic slide.
3. Dispense 1 drop (17 uL) of antigen suspension onto the same ring using a syringe.
4. Place the slide onto a rotator for 4 minutes at 180 rpm.
5. Immediately read the slide at 10X objective under the microscope.
Quantitative test
1. Dispense 50 uL of 0.9% saline onto 5 separate ceramic rings.
2. Transfer 50 uL of undiluted serum into the first ring to make a 1:2 dilution and mix the solution.
3. Transfer 50 uL of diluted serum into the second ring to make a 1:4 dilution and mix the solution.
4. Repeat this step for the 3rd (1:8), 4th (1:16) and 5th (1:32) ring.
5. Add 1 drop of antigen suspension into all the rings.
6. Place the slide onto a rotator for 4 minutes at 180 rpm.
7. Immediately read the slide at 10X objective under the microscope.
Results:
Qualitative test
Medium/ large clumps - Reactive (R)
Small clumps - Weakly reactive (WR)
No clumping/ very slight roughness - Non-reactive (NR)
Quantitative test
- The last dilution titre that produces a reactive result will be reported.
- E.g If 1:16 is weakly reactive and 1:8 is reactive, 1:8 dilution will be reported.
Treponemal test
For treponemal tests, instead of non-specific antigens being used, antigens specific for T. pallidum is used to detect antibodies against T. pallidum. These tests are used as a confirmatory test and can detect all stages of syphilis, excluding the first 3 to 4 weeks after initial infection, as humoral antibodies specific for T. pallidum have not been produced. In secondary, latent and congenital syphilis, the treponemal tests would be 100% reactive.
However, these treponemal tests cannot be used to determine the efficacy of treatment or the presence of a re-infection as the tests are qualitative tests.
One of the treponemal tests carried out in the lab include the Treponema pallidum particle agglutination assay (TPPA).
Treponema pallidum particle agglutination assay (TPPA)
Principle:
The TPPA test is used to detect antibodies specific to treponemas, which causes syphilis. For this test, coloured gelatin particles carriers sensitized by T. pallidum antigens are used. So the patient’s serum is first diluted with sample diluent in microplate wells. Then, the sensitized gelatin particles will be added to the diluted serum. If antibodies specific to T. pallidum are present, it will bind to the particles, forming a smooth mat of agglutinated particles. For non-agglutinated particles, they will gather to form a button at the bottom of the wells. [8] This will indicate a negative test. As a control to ensure non-specific reactions have not occurred, unsensitized cells will also be used. If there is agglutination with the unsensitized cells, it means that there is non-specific agglutination, thus rendering the test as not accurate.
Procedure:
1. Label 4 wells on the microtitre plate wells 1 to 4.
2. Add 100 uL of sample diluent (provided by the test kit) into well 1 and 25 uL into wells 2, 3 and 4.
3. Add 25 uL of serum into the first well and mix thoroughly.
4. Transfer 25 uL of diluted serum from the first well to well 2 and mix thoroughly.
5. Repeat this step for wells 3 and 4 and discard the last 25 uL taken from well 4.
6. Add 25 uL of sensitized cells into well 3 and 25 uL of unsensitized cells into well 4.
7. Place the plate on an automatic shaker for a few seconds and incubate the plate at room temperature for 2 hours.
Results:
- Particles settled in the centre of the well in the shape of a button: Non-reactive (-)
- Particles concentrate in the shape of a compact ring with a smooth round outer margin: Indeterminate (±)
- Definite large ring with a rough multiform outer margin and peripheral agglutination: Reactive (+)
- Agglutinate particles spread out covering the bottom of the well uniformly: Reactive (++)
Anyway, for the past 2 weeks, I have been attached to the Serology department. Within this department, there are 2 labs: STD lab and Serology lab. In the STD lab, tests for syphilis and gonorrhoeae are done. But I think the gonorrhoeae section will be moved to the Bacteriology department at the beginning of November. In the serology lab, tests for autoimmune and infectious disease are done. But for this post, I shall just concentrate on the tests for syphilis.
Let me give a brief introduction to syphilis. I am sure you all know that syphilis is a sexually transmitted disease. It is caused by bacteria known as Treponema pallidum. This organism belongs to the order Spirochaetales and is a member of the Treponemataceae family. It is a thin, delicate organism with tapering ends. Its size can vary from 6 to 15 microns in length with a thickness of around 0.25 microns. The organism also has 6 to 14 spirals with pointed ends that have finely spiral terminal filaments. Syphilis can be divided into several stages: early syphilis, late syphilis and congenital syphilis. Early syphilis is the initial stage of infection while late syphilis occurs only 10 to 20 years after initial infections. Congenital syphilis is not connected to early or late syphilis. It is syphilis infection in infants.
Tests for syphilis can be divided into non-treponemal and treponemal tests.
Non-treponemal test
Non-treponemal tests are screening tests as they screen patients for non-specific reagin antibodies that are produced during an infection. The antigens used for screen are lecithin and cardiolipin. When treponemas is present, the lipid from the cell surfaces would cause the host to produce anti-lipid IgG and IgM antibodies. So the lecithin and cardiolipin antigens will bind to these antibodies, forming complexes, which will stay suspended in the tests via flocculation. The type of non-treponemal test used in the lab is the Venereal disease research laboratory (VDRL) test.
Venereal disease research laboratory (VDRL) test
Principle:
VDRL test is a slide microflocculation test for syphilis. When treponemas is present in the host, anti-lipid IgG and IgM antibodies known as reagin will be produced, in response to the lipid on the treponemal surface. These antibodies will then react with substances on the mitochondrial membrane. The antigen used in this test is mixed with buffered saline and consists of cardiolipin, cholesterol and lecithin. These substances will react with the reagin present in the serum of syphilitic patient.
A reactive test means that flocculation, a combination of principles of precipitation and agglutination, has occurred when the antigen reacts with antibody, thus forming antigen-antibody complexes. These complexes can be viewed as big clumps under the microscope. A weakly reactive test means that the clumps are smaller than that of a reactive test. A non-reactive test means that no clumps are observed under the microscope.
The test is divided into qualitative test and quantitative test. For qualitative test, it just indicates whether the sample is reactive or not. For the quantitative test, dilution of the serum will be done. So the highest dilution with a reactive result can be known.
However, as reagins may be produced in other treponemal diseases and in response to a non-treponemal disease, which results in tissue damage, a reactive VDRL test does not confirm a T. pallidum infection resulting in syphilis. But for a reactive VDRL test, a treponemal test (e.g TPPA) would follow to confirm the reactive result.
Procedure:
Qualitative test
1. Spin the blood at 3000 rpm for 10 minutes.
2. Transfer 50 uL of serum onto 1 ring of the ceramic slide.
3. Dispense 1 drop (17 uL) of antigen suspension onto the same ring using a syringe.
4. Place the slide onto a rotator for 4 minutes at 180 rpm.
5. Immediately read the slide at 10X objective under the microscope.
Quantitative test
1. Dispense 50 uL of 0.9% saline onto 5 separate ceramic rings.
2. Transfer 50 uL of undiluted serum into the first ring to make a 1:2 dilution and mix the solution.
3. Transfer 50 uL of diluted serum into the second ring to make a 1:4 dilution and mix the solution.
4. Repeat this step for the 3rd (1:8), 4th (1:16) and 5th (1:32) ring.
5. Add 1 drop of antigen suspension into all the rings.
6. Place the slide onto a rotator for 4 minutes at 180 rpm.
7. Immediately read the slide at 10X objective under the microscope.
Results:
Qualitative test
Medium/ large clumps - Reactive (R)
Small clumps - Weakly reactive (WR)
No clumping/ very slight roughness - Non-reactive (NR)
Quantitative test
- The last dilution titre that produces a reactive result will be reported.
- E.g If 1:16 is weakly reactive and 1:8 is reactive, 1:8 dilution will be reported.
Treponemal test
For treponemal tests, instead of non-specific antigens being used, antigens specific for T. pallidum is used to detect antibodies against T. pallidum. These tests are used as a confirmatory test and can detect all stages of syphilis, excluding the first 3 to 4 weeks after initial infection, as humoral antibodies specific for T. pallidum have not been produced. In secondary, latent and congenital syphilis, the treponemal tests would be 100% reactive.
However, these treponemal tests cannot be used to determine the efficacy of treatment or the presence of a re-infection as the tests are qualitative tests.
One of the treponemal tests carried out in the lab include the Treponema pallidum particle agglutination assay (TPPA).
Treponema pallidum particle agglutination assay (TPPA)
Principle:
The TPPA test is used to detect antibodies specific to treponemas, which causes syphilis. For this test, coloured gelatin particles carriers sensitized by T. pallidum antigens are used. So the patient’s serum is first diluted with sample diluent in microplate wells. Then, the sensitized gelatin particles will be added to the diluted serum. If antibodies specific to T. pallidum are present, it will bind to the particles, forming a smooth mat of agglutinated particles. For non-agglutinated particles, they will gather to form a button at the bottom of the wells. [8] This will indicate a negative test. As a control to ensure non-specific reactions have not occurred, unsensitized cells will also be used. If there is agglutination with the unsensitized cells, it means that there is non-specific agglutination, thus rendering the test as not accurate.
Procedure:
1. Label 4 wells on the microtitre plate wells 1 to 4.
2. Add 100 uL of sample diluent (provided by the test kit) into well 1 and 25 uL into wells 2, 3 and 4.
3. Add 25 uL of serum into the first well and mix thoroughly.
4. Transfer 25 uL of diluted serum from the first well to well 2 and mix thoroughly.
5. Repeat this step for wells 3 and 4 and discard the last 25 uL taken from well 4.
6. Add 25 uL of sensitized cells into well 3 and 25 uL of unsensitized cells into well 4.
7. Place the plate on an automatic shaker for a few seconds and incubate the plate at room temperature for 2 hours.
Results:
- Particles settled in the centre of the well in the shape of a button: Non-reactive (-)
- Particles concentrate in the shape of a compact ring with a smooth round outer margin: Indeterminate (±)
- Definite large ring with a rough multiform outer margin and peripheral agglutination: Reactive (+)
- Agglutinate particles spread out covering the bottom of the well uniformly: Reactive (++)
The results
So, this is a description of what I did in the STD laboratory. Hope that this post has been ‘understandable’.
5 more weeks to go. So let’s carry on working hard for our SIP and MP =).
- Li Ping-
0607498C
TG o2
5 comments:
Hi liping
I guess our jobs now are quite similar. I handle Pap smears while you look out for STD in the blood. Look at my post too.
My question is. How long after the syphyllis infection occurs then you will be able to detect the antigen. In other words, from the time of the bacteria entering, to the time of showing it in the test, how long does it take?
For example, I believe HIV test, you need to repeat it after 3 to 6 months again because the antibody doesn't show at the early stages of infection.
Thank you
Ernest
0606330i
week 16
hey..
wat a post you have here.
i ordered TPPA test occasionally here in my lab. i have always wondered what test is that as it is not done in my lab. Its a send out test for our case. im glad you shared this.
tanx..
sutiana
Answers =)!
To Ernest:
3 weeks after the initial infection, the primary stage ulcers will appear. These ulcer will resolve within 1 to 5 weeks. The humoral antibodies, that are used for the test, will only develop 1 to 4 weeks after the ulcer has formed.
So err, to answer your question, it will take around 3 months after the initial infection for the antibodies to be detected.
Btw, for this test, we want to detect the presence of antibodies, not the antigens =).
Hope it answers your question!
To Suitiana:
I'm glad that i am able to help shed some light on this test =).
-Li Ping-
TG o2
Hi Li Ping =)
Liyana here, from group 8.
I am curious, why are gelatin particles used for the TPPA test? I mean, is there a special relationship between T. pallidum antigen-gelatin-antibodies?
Oh! wait, it's gelatin particles carriers.. meaning cells that carry gelatin particles, right? wow.. what sort of cells are they?
-----
Nor Liyana
0607927A
TG02 - Group 8
Hello Liyana!
ehh.. it could be my improper phrasing that led to some confusion. Gelatin particles are not cells. They are just certain materials that are able to carry the T. pallidum antigents. So gelatin particles are used simply because they can be sensitized with antigens for the detection of antibodies.
err. hope i've answered your question =).
Li Ping
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