Diaskintest

The causative agent of tuberculosis (Возбудитель туберкулеза)

Human Tuberculosis is caused by pathogenic strains of Mycobacterium tuberculosis (MBT),
mainly Mycobacterium Tuberculosis and Mycobacterium Bovis. They are able to penetrate, live
and proliferate in the human body due to the main species trait - virulence. The degree of
virulence can carry, increase or decrease under the influence of environmental factors and the
condition of the microorganism.
Virulence is determined primarily by the genetic structure of Mycobacteria. The structure of the
genome of pathogenic strains of M. Tuberculosis and M. Bovis is 99.9% identical and contains
approximately 4000 genes. The genome of mycobacteria contains a specific RD1 region. In this
region, pathogenic mycobacteria encode the synthesis of specific proteins, CFP10 and ESAT6,
which are produced dye to the active proliferation of mycobacteria in the human body and
determines their virulence properties.

Formation of anti-tuberculosis immunity.

To prevent the development of severe forms of tuberculosis in children in our country, newborns
are vaccinated on days 3-7 of life. The vaccine contains live strains of M.Bovis (BCG). In
contrast virulent MBT strains, the M.Bovis (BCG) vaccine strain does not contain a fragment of
the RD1 genome that encodes the secretion of CFP10 and ESAT6 proteins, which determine the
main virulent properties of the infectious agent. As a result of vaccination, a long-term specific
immunity against tuberculosis is formed up to under or over 7 years of age.

Contamination .

A TB patient with an active form of the disease releases MBT into the environment when
coughing, sneezing and talking. Most often, contamination occurs by inhalation of air that
contains pathogenic MBT. Insufficient activity of the mucociliary clearance of the upper
respiratory tract predetermines the preconditions for the penetration of mycobacteria into the
trachea, bronchi and, ultimately, the end sections of the bronchioles - the alveoli. From the
alveoli MBT easily penetrate into the lymphatic vessels and spread through the body with the
flow of lymph. The most frequent sdementationsdementation area and subsequent deposition of
infectious agent in the human organsism are the intrathoracic lymph nodes, less often the lungs
and other organs.

Cell immunity.

When a person comes into contact with a virulent strain of MBT, human sensibilization occur to
the infectious agent and the reaction of cell mediated anti-tuberculosis immunity is formed.

Sensibilization. Сенсибилизация.
In the zone of localization of the pathogen, macrophages actively absorb MBT (the process of
phagocytosis), where their degradation and death occurs. Specific antigens of MBT, and in
particular the CFP10 and ESAT6 proteins, which determine the virulence properties of the
infectious agent, approach the surface of the macrophage plasma membrane for presentation to
T-lymphocytes. The macrophage secretes anti-inflammatory cytokines, interleukins that attract
T-lymphocytes to the focus of infection. They read information about antigens from the
macrophage membrane and turn into memory cells.

Delayed type hypersensitivity reaction (or hypersensitivity inflammatory reaction)

Activated T-lymphocytes produce the effector cytokines interferon gamma (IFN-ϒ) and tumor
necrosis factor-alpha (TNF-α). They attract a large amount of different cells from the capillaries
to the site of infection: macrophages, lymphoid cells, granulocytes and others. These cells are
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involved in the formation of granulomas and thus inhibit the distribution of Mycobacterium
tuberculosis in the organism .

Development of tuberculosis process

The outcome of primary contamination from MBT depends on mycobacterial virulence, intensity
and duration of bacterial load and the human organism and its immunity. Accordingly,
tuberculosis infection occurs in one of three directions: -

1st variant: in 90-95% of cases the pathological process does not develops in persons with
adequate immune response, and moderate bacterial load after contamination with MBT. After
granuloma formation in macrophages intensive death of MBT occurs with subsequent apoptosis
of macrophages, in the central part of granuloma caseous necrosis forms around the site of
infection a dense fibrous membrane capsule forms which restricts the infected area from adjacent
healthy tissues. The disease does not develop.

2nd Variant: the mycobacterium population escapes from toxic effect of macrophages and
preserves within the low activity. “Dormant” sleeping condition of mycobacterial, in such
conditions disease does not develops but the risk remains of getting sick in the future when a
human immune system is weakened being exposed to HIV infection, treatment with
immunodepressants, treatment with tumor necrosis factor alpha-blockers, severe chronic stress,
severe diseases, starvation, infected person with high disease risk which have not had
chemoprophylaxis activate dormant sleeping mycobacterium which predetermines the disease
development.

3rd Variant: the organism 5-10% infected people which have large bacterial load, active
proliferation and increased mycobacterial virulence as well as immune deficiency is not able to
manage infection which results in development of disease.

Mantoux test.

In our country, for almost 100 years, tuberculin diagnostics made world wide, the intradermal
Mantoux test, using tuberculin to determine the specific sensibilization of the body to MBT.

Tuberculin is a culture filtrate which contains a large number of mycobacterial antigens. They
are inherent of both tuberculous and non-tuberculous, and vaccine strains of mycobacteria’s. As
a result of primary immunization with BCG vaccination of contamination with mycobacterium
tuberculosis, mature memory T-cells accumulate in the organism, which keeps the information
about the antigenic properties of mycobacteria with which they already are familiar with.

In response to introduction (injection) of tuberculin, when setting up an intradermal Mantoux

test, the process of an immune reaction is triggered by the type of delayed-type hypersensitivity.

A positive Mantoux reaction develops: with BCG vaccination, sensibilization with non-
tuberculous mycobacteria, contamination with tuberculosis Mycobacteria. Among people who
test positive for the Tuberculin Mantoux Test, it can be hard for physicians to interpret the results
of the test and decide whether additional testing and prescribing preventive treatment is
necessary.

Low specificity and a large percentage of false positive reactions during the Mantoux test result
to hyperdiagnosis of the disease and unreasonable prescribing of preventive therapy. At the same

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time, meanwhilefor a positive reaction to be regarded as post-vaccination in the presence of an
active tuberculosis infection and a high risk of developing the disease.

Test systems for Interferon-gamma release assays in-vitro (IGRA)

The results of international studies which aimed to interpret mycobacterium genome and detect
region RD1 specific for strains of Mycobacterium Tuberculosis and Mycobacterium Bovis have
lead to the development of two variants of diagnostic test systems which are called test systems
for interferon gamma release assay (IGRA), such test systems are based on laboratory evaluation
interferon gamma release by activated memory t-cells in response to stimulation with
recombinant protein ESAT6 and CFP10, fresh blood which is taken from study subject is
required for the analysis . System ELISPOT (T-SPOT.TB)- i(solation of peripheral blood
mononuclear cells)evaluates visually or with devices with the number of stains zones, I
sensiblize peripheral t-cells which release interferon gamma after incubation with recombinant
protein CFp10, ESAT6.
System ELISA quantifier coat TB golden tube evaluates concentration of interferon gamma in
blood plasma with enzyme linked immunosorbent analysis after incubation of whole blood with
recombinant protein CFP10, ESAT6, quantiFERON TB golden in tube. IGRA test system have a
wide range of disadvantages that prevents their use in population screening test for tuberculosis
in particular high cost, necessary equipped laboratory and specialists, special strict requirements
of biological material preparation for the test storage conditions and precise blood processing as
well as required intravenous manipulation in children as so far IGRA test systems requires
further studies to define their relevance in TB diagnostics.

Diaskintest

The developments of Russian scientists in recent years have made it possible to improve the
quality of diagnosis of tuberculosis infection due to the creation of an innovative intradermal test
Diaskintest. It is made with a recombinant protein, which is a combination of CFP10 and
ESAT6, which are essential proteins that determine the virulence properties of MBT and are
formed during their active proliferation in the organism.

These proteins are not synthesized by the M.Bovis (BCG) vaccine strain, as well as by most
nontuberculous mycobacteria. Therefore, in contrast to tuberculin, Diaskintest does not give a
cross-reaction on the antigens of the BCG vaccine strain and most non-tuberculous
mycobacteria. The setting of Diaskintest is identical to the Mantoux Test and does not require
serious skills and material expenses. The result of the reaction is evaluated in 72 hours. The
action of the drug is based on the development of an immune response of the type of delayed-
type hypersensitivity.

Diaskintest give positive reaction with of papules of any size, with an active tuberculosis
infection - the proliferation of mycobacteria in individuals with an adequate immune response to
specific antigens CFP 10 and ESAT6 in persons with high disease risk and at early stage of
active tuberculosis infection.

Due to the high almost one hundred percent specificity, Diaskintest usually does not give false
positive reactions in relation with BCG vaccination and sensibilization by non-tuberculous
mycobacteria. It allows to diagnose with high accuracy the active tuberculosis process, as well as
infected individuals with a high risk of developing the disease.