Screening and Quantification of Treponema pallidum using TPHA Test

This report details the process and results of a Treponema pallidum haemagglutination (TPHA) test, a serological method used to detect antibodies against the syphilis-causing bacterium. The study involved screening and quantification phases using a TPHA test kit, including negative and positive con...Read More

Running head: SCREENING AND QUANTIFICATION OF TRIPODEMA PALLIDUM IN
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SCREENING AND QUANTIFICATION OFTRIPODEMA PALLIDUM IN SERUM USING
THE TPHA TEST.

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Objectives
To investigate the presence of Treponema pallidum in the patient serum
To find out how the Treponema pallidum haemagglutination test usually works.
Introduction
Medical research has come a long way over the years. From medieval treatment
methodologies, which made it impossible to catch an infection in its infancy and therefore
find a remedy to it early enough, research has improved significantly. New methods are
discovered everyday, which simplifies the research processes. The discovery of novel
methodologies, organisms, infections, research technologies, etc has however, always been
either in response to a calamity or accidentally by discovering a solution to a problem that at
the time, was not a concern. For instance, the discovery of penicillin by Doctor Alexender
Fleming in 1928, was purely unintended (Gaynes, R. 2017). The invention of the pacemaker
also happened by chance since the inventor was trying to develop a device that could record
the rhythm of the heart. Because of this fact, medical research is a very risky undertaking.
The laboratory is home to many, very dangerous chemicals, study organisms and

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microorganisms that are on their own harmful to humans, or can secrete chemicals that are
detrimental to the health of a person. As a scientist, one’s health is constantly in danger from
both known and unknown sources. For this reason, scientists have developed ways of
protecting themselves from infections and other dangers that they are usually exposed to
while in the field.Scientific research facilities must have proper clothing that protect the
scientist including gloves, lab coats face masks, eye goggles, etc. which must be worn by the
researcher while in the laboratory. Other than protective gear, personal hygiene is of utmost
importance in any research facility. Washing one’s hands, safe disposal of wastes and
handling of sharp objects such as scalpels and needles, proper spillage management, and
proper methods for surface decontamination must be strictly followed to prevent
contamination and infections.
Man has however managed to develop ways and kits for screening and identification of most
of the infection causing pathogens. In case of an infection or infection scare, it is imperative
to pinpoint the causative agent as quickly as possible, so as to monitor the infection properly
and even come up with a remedy in time, hence the development of kits that can give the
intended result within as little time as possible. A blood examination is one of the most
frequently used methods that are simple to carry out and take the shortest time possible to
carry out. Other methods include microbial culture of body fluid/swabs in the laboratory and
thereafter carrying out a test such as the Gram and Acid Fast differentiation tests in order to
categorize the pathogens, identification of pathogen DNA/RNA for instance by polymerase
chain reaction (PCR), identification of pathogen antigens, and identification of anti-pathogen
antibodies by Enzyme-Linked Immunosorbent Assay (ELISA).
Treponema pallidum is a spiral-shaped bacterium (spirochete) that causes syphilis (Radolf et
al., 2016,). The bacterium stains weakly negative in the Gram test (Sekikawa, Oda, Hongo
and Ono, 2017). Syphilis is considered a sexually transmitted disease because it is commonly

SCREENING AND QUANTIFICATION OFTRIPODEMA PALLIDUM IN SERUM USING
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transmitted through sexual intercourse, though it can be transmitted to a healthy person by
close body contact with an infected person, or from an infected mother to her unborn child.
The history of syphilis goes back to as far as the Second World War, during the infamous
Tuskegee syphilis trials, where Africans living in Tuskegee were subjected to mandatory
trials by the United States government. Research has however shown that the disease cannot
spread through the sharing of normal things like a doorknob. Syphilis is treatable if it is
discovered in its early stages. If left untreated, however, it can lead to complications such as
damage to organs such as the heart, bones, liver, blood vessels, neurological disorders and
eventually death (Qadir and Fatima, 2019). According to Qadir and Fatima (2019), the
disease fast depicts itself as a painless sore, called a chancre, usually on the genitals, mouth
or rectum. The chancres appear about three weeks post-exposure. This is usually the first
stage of infection. Research has shown that the disease undergoes four stages, with each
having more severe symptoms than the one before. The stages include the primary,
secondary, latent, and tertiary stages (Sivakumar and Banupriya, 2017). Without treatment,
the disease enters the next phase which is characterized fever, sore throat, muscle aches,
patchy hair loss and swelling of lymph nodes. Other symptoms that manifest in the later
stages include dementia, abnormal gait, headache, and weakness. In children, the symptoms
include saddle nose, blisters on the hands and feet, and fever. Upon early discovery, the
disease can be completely cured by penicillin (Kingston et al., 2016). The signs and
symptoms of syphilis are diverse and not entirely specific to it (Forrestel, Kovarik, and Katz,
2019). It is therefore mandatory that the best laboratory equipment and qualified personnel be
involved in its diagnosis.
There are several tests that can be carried out to determine whether Treponema pallidum is
present in a test sample or not. They include darkfield microscopy of samples and serological
tests, which include both treponemal and non-treponemal tests. The typical non-treponemal

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test that is in use is the venereal disease research laboratory (VDRL) slide test, which has
been used in India the most (Santhi et al., 2016). The venereal disease research laboratory
slide test, according to Santhi et al., (2016), is unreliable since it gives false positives and
false negatives, and is also less responsive in primary and late stages of the infection. The
tests that have been deemed specific tests for syphilis are; fluorescent treponemal antibody
absorption (FTA-ABS) test, the FTA-ABS DS (double staining), the microhaemagglutination
assay for Treponema pallidum (MHA-TP), and the haemagglutinationation Treponemal test
for syphilis (HATTS).
The recommended test for determining the existence of Treponema pallidum in the laboratory
is called the Treponema pallidum haemagglutination test (TPHA test). It is a simple test to
perform and very convenient. It is also more sensitive than the VDRL and FTA-ABS tests in
the early and late stages of the disease.in this experiment, the TPHA test for syphilis was
used.
Principle
The TPHA test employs the principle of passive haemagglutination, by detecting
immunoglobulins, specifically the IgG and IgM antibodies, which are produced against
Treponema pallidum. In this reaction, the serum containing the anti-treponemal antibodies
reacts with avian erythrocytes which had previously been sensitized with sonicated
Treponema pallidum (Nichol’s strain). A positive test is depicted by the agglutination of cells
to form a smooth mat-like mass of cells. The formation of a compact button, on the other
hand, is an indication of the absence of a reaction, thus the avian erythrocytes settle at the
bottom of the plate, forming the button. The control tests (avian erythrocytes that are devoid
of Treponema pallidum antigens) are used to detect any non-specific reactions

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Materials
Oxoid Microbiology Products TPHA Test Kit (Code DR0530)
Patient Samples
Methodology
Two sets of tests were carried out, a screening test and a quantitation test, and clean 96 well
microtitre plates used, one for each test.
Screening Plate
For the screening test, the following criteria were followed in dispensing the TPHA diluent
into the microplate wells; into wells labeled A1, C1 and E1, 25 l𝛍 of the TPHA diluent was
pipetted. Into the wells labeled A2, C2, and E2, 100 l were added, to wells A3, C3, and E3,𝛍
A4, C4 and E4, 25 l of the TPHA diluent was added respectively.𝛍
Negative control
The negative control was prepared as follows; to well A1, containing 25 l of the TPHA𝛍
diluent, an equal amount of the negative control serum was pipetted and thoroughly mixed
with the TPHA diluent. 25 l of the solution was then drawn from A1 and diluted into A2𝛍
which was then thoroughly mixed. 25 l of solution A2 was then drawn as with A1 and𝛍
dispensed into well A3, which was then mixed as before. The 25 l drawn from this well was𝛍
then discarded. Another 25 l was drawn from well A2 and added to the TPHA diluent in𝛍
well A4 and the solution thoroughly mixed as with the other wells. Afterward, 25 l from𝛍
solution A4 was drawn and discarded into a virkon waste.

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Positive control
Into well C1, containing 25 l of the TPHA diluent, an equal amount of the positive control𝛍
serum was added and well mixed. 25 l of solution C1 was then drawn and added to well C2.𝛍
The solution was thoroughly mixed. 25 l of solution C2 was then drawn and added to well𝛍
C3, mixed well and then 25 l of solution C3 drawn and discarded into a Virkon waste.𝛍
Another 25 l was taken from well C2 and dispensed into well C4. After thoroughly mixing,𝛍
25 l of solution C4 was drawn and discarded into the Virkon waste.𝛍
Patient serum
25 l of the sample serum was dispensed into the microwell E1. It was then mixed five times𝛍
using a micropipette. 25 l of the mixed solution E1 was then drawn and added into well E2,𝛍
which was well mixed and the same amount of solution E2 transferred into E3. After mixing
the contents of well E3, 25 l was drawn and thrown into the Virkon waste.𝛍
Quantitation Plate
The quantitation test was carried out on the second microtitre plate. Into microwells A1 to
A6, 25 l of the TPHA diluent was pipetted. Solution A2 of the screening plate was𝛍
intermixed once again, and 25 l of it pipetted into well A1 of the quantification plate. The𝛍
solution was mixed rigorously, 25 l of it drawn and disposed of into a Virkon pot. 25 l of𝛍 𝛍
the properly mixed solution C2 from the screening plate was transferred into well A2 of the
quantitation plate and thoroughly mixed using a micropipette. 25 l of this solution was then𝛍
drawn from this well and then pipetted into well A3, which was then mixed rigorously using
a micropipette. The procedure was then repeated in the rest of the wells. The 25 l drawn𝛍
from well A6 was then discarded. 75 l of the test cells was then added to wells A4, C4 and𝛍

SCREENING AND QUANTIFICATION OFTRIPODEMA PALLIDUM IN SERUM USING
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E4 of the screening plate, and wells A2 to A6 of the quantification plate. To wells A3, C3 and
E3 of the screening plate and well A1 of the quantification plate, 75 l of control cells were𝛍
added. The solutions were incubated at 250C for a period of one hour. The plates were then
taken and inspected for precipitation and the results noted down.
Results
screening plate
Wells A1, A2, C1, C2, E1, and E2 did not have agglutinations or compact buttons. In
wells A3 and A4, compact buttons were observed. In wells C3 and E3, buttons are
observed though they are not as compact as in A3, and A4. In wells C4 and E4 there is
agglutination
Quantitation Plate
In well A1, a compact button was observed. In wells A2, A3, A4, A5, and A6
agglutinations were observed, which showed a diminishing trend from A2 to A6.

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Discussions
As stated earlier, the Treponema pallidum haemagglutination test detects the presence of T.
pallidum in a sample through a reaction between antibodies in the sample serum that has been
raised against the antigens (T. pallidum) and the antigens coated on the avian cells. Positive
and negative results are shown an agglutination reaction of specific characteristics and a
compact button respectively.
In the screening plate, A3 and A4 gave a negative answer. These wells were the negative
controls, that is, they contained avian erythrocytes not coated with the antigen. Therefore,
there was no antibody-antigen reaction hence the formation of a compact button. In wells, C3
and C4, and E3 and E4 showed the presence of antibody-antigen reaction. Wells C3 and C4
were the positive controls, and therefore contained human antibodies raised against
Treponema pallidum. The antibodies in this control reacted with the antigens on the test cells
resulting in agglutination. The patient serum (wells E3 and E4) also had a positive reaction,
which resulted in agglutination. Wells C4 and E4 showed more reaction to the antigen than
wells C3 and E3 whereas all the wells in columns 1 and 2 appeared empty. This trend can be

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attributed to the serial dilution-like reaction of the antibodies and antigens, where the
agglutination is doubled as the sample is added from one well to the next.
The quantitation plate showed a different trend. In well A1, the negative control is added
hence there is no reaction. In wells A2 to A6 on the other hand, there is an observable
decreasing trend with the number of wells. This declining trend can be attributed to the serial
dilution that was carried out using solutions from screening well C2.
Conclusion
The Treponema pallidum haemagglutination test is a quick technique that is quite easy to use.
This technique is time-saving as the whole process cannot take more than three hours. It is
also a very reliable method for both qualitative and quantitative studies of the bacterium
Treponema pallidum since it is depended on the reaction between the antigens and the anti-
treponemal antibodies. It is therefore very specific.

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