OBJECTIVES Up on completion to this presentation you will be able to: know about H pylori definition and background Know diagnostic method of H pylori List available serological test for H pylori with their:- Intended use Principle Precaution Limitation Interpretation and Quality Control Introduction Most common chronic bacterial infection in world. Key constituent of human micro biome. Gram negative Spiral shape Multiple unipolar flagella – moves freely Produces urease Micro-aerophillic 3 μm long with a diameter of about 0.5 μm More common in low socioeconomic status Humans are major reservoir Housing density, crowded conditions in the home, number of siblings, sharing a bed, and lack of hot running water

1. Helicobacter pylori
SEROLOGICAL AND
OTHER LAB DIAGNOSIS
Yonatan Demeke
Jimma, Ethiopia
AUG, 2022
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2. OBJECTIVES
Up on completion to this presentation you will
be able to:
 know about H pylori definition and background
 Know diagnostic method of H pylori
 List available serological test for H pylori with
their:-
Intended use
Principle
Precaution
Limitation
Interpretation and
Quality Control
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4. INTRODUCTION
 Most common chronic bacterial infection in world.
Key constituent of human micro biome.
 Gram negative Spiral shape
 Multiple unipolar flagella – moves freely
 Produces urease
 Micro-aerophillic
 3 μm long with a diameter of about 0.5 μm
 More common in low socioeconomic status
 Humans are major reservoir Housing density,
crowded conditions in the home, number of
siblings, sharing a bed, and lack of hot running
water
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5. TRANSMISSION
 Gastro oral route
 Feco oral route
 Oral - Oral route Person to person transmission
 Developing countries Contaminated water
 Inadequately disinfected endoscopic devices
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7. PATHOGENESIS
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9. Diagnosis of H. Pylori
INVASIVE
 Histopathology
 Rapid Urease Test
 Culture
 PCR
NON INVASIVE
 Urea Breath Test
 Serology
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10. Invasive Diagnosis Techniques
 The most specific test to detect H pylori infection
is culture, but the sensitivity is usually lower than other
methods because the organism is not evenly
distributed throughout the gastric tissue.
Endoscopic diagnostic tests
 Biopsies taken at endoscopy are most
commonly for histological analysis and urease testing.
Rapid urease test (CLO test):
Biopsies of gastric mucosa are placed in a gel containing
urea, and the subsequent ammonia production causes a pH
change, which is observed as a color change
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12. Non-invasive techniques
Urea Breath test
This test examines your breath for the
presence of Helicobacter pylori bacteria, which
can cause gastritis (inflammation of the mucous
membrane of the stomach) or ulcers in the
stomach and small intestine.
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14. SEROLOGICAL TESTS
1. Detection of H pylori antigen
 H. pylori Antigen ELISA Test Kit
 Rapid Antigen test
2. Detection of specific anti-H pylori IgG antibodies
 Helicobacter pylori IgG ELISA Kit
 rapid detection of Antibodies to H. pylori
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15. 1. H. pylori Antigen ELISA Test
Kit
INTENDED USE
It is intended as an aid in the diagnosis of possible H. pylori
infection and in the follow-up of patients undergoing
antimicrobial
therapy.
PRINCIPLE OF THE TEST
 The H. pylori Antigen ELISA Test Kit is a solid phase
enzyme immunoassay based on sandwich principle for the
qualitative and quantitative detection of H. pylori antigen in
human stool.
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16.  During testing, the antigens are extracted from the
specimen with extraction solution and added onto the
antibodies coated micro-well plate along with the
enzyme- conjugated antibodies to H. pylori, and then
incubated.
 If specimens contain H. pylori antigens, it will bind to the
antibodies coated on the micro-well plate and
simultaneously bind to the conjugate to form immobilized
antibody-H. pylori antigen-conjugate complexes.
 If specimens do not contain H pylori antigens, the
complexes will not be formed.
 After initial incubation, the micro-well plate is washed to
remove unbound materials.
 Substrate A and substrate B are added and then
incubated to produce a blue color indicating the amount
of H. pylori antigens present in the specimens.
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17.  Sulfuric acid solution is added to the micro-well plate to
stop the reaction producing a color change from blue to
yellow.
 The color intensity, which corresponds to the amount of
H. pylori antigens present in the specimens, is measured
with a microplate reader at 450/630-700 nm or 450 nm.
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18. PRECAUTIONS
 Follow the instructions for use carefully.
 Wear protective clothing and disposable gloves when
dealing with samples and reagents. Wash hands after
operations
 Do not use reagents beyond the labeled expiry date.
 Do not mix or use components from kits with different
batch codes.
 Avoid cross contamination between reagents to ensure
valid test results.
 Follow the wash procedure to ensure optimum assay
performance
 Use a new pipette tip for each specimen assayed.
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19. SPECIFIC MATERIALS AND REAGENT
USED
1. H Pylori Antibody Micro-
well Plate
2. H pylori Antibody
Conjugate
3. Concentrated Wash
Buffer (25x)
4. Substrate A and B
5. Stop Solution
6. Freshly distilled or
deionized water.
7. Sodium hypochlorite
solution for
decontamination.
8. Disposable gloves.
9. Automated processor
10. Vortex mixer for
specimen mixing
(optional).
11. Disposable reagent
reservoirs.
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21. COLOR CODES
OF REAGENTS
Blue-sample dilution
Yellow-for conjugate
dilution
- Brown-Substrate
- Blue cap -negative
control.
- Red- positive control
Transparent-
Concentrated wash
solution
Transparent with red
cap -stop solution
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22. STORAGE AND STABILITY
 Components of the kit will remain stable through the
expiration date indicated on the label and package when
stored between 2-8°C
 Place unused wells in the zip-lock aluminum foiled
pouch and return to 2-8 °C, under which conditions the
wells will remain stable for 3 months from the opening
date.
 Do not expose reagents especially the Substrate to
strong light or hypochlorite fumes during storage or
incubation steps.
 Do not store Stop Solution in a shallow dish or return it
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23. SPECIMEN COLLECTION AND
PREPARATION
 Stool specimen is needed to check presence of H pylori
Antigen by H. pylori Antigen ELISA Test Kit
 Stool samples should be collected in clean containers.
 Samples can be stored in the refrigerator (2-8 °C) for 1-
2 days prior to testing.
 For longer storage, the specimen must be kept frozen
at -20ºC.
In this case, the sample should be totally thawed and
brought to room temperature before testing.
 The patient has to be asked to collect the specimen
avoiding any possible contact with urine or water.
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24.  The patient submitted to the test should not be under
antibiotic or anti-bacterial treatments as this
pharmaceutical therapy is known to affect H. pylori up to
a certain extent, depending on the antibiotic used, giving
rise to false interpretation.
 If specimens are to be shipped, they should be packed in
compliance with local regulations covering the
transportation of etiologic agents.
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25. PROCEDURE FOR THE ASSAY
1. Remove unused strips from the micro-well plate,
and store in the original resealable pouch at 2-
8°C.
2. Allow reagents and specimens to reach room
temperature (15- 30°C) prior to testing.
WASH PROCEDURE
a) The wash procedure is critical. Insufficient
washing will Result in a poor precision and falsely
elevated absorbance readings.
b) Prepare working wash buffer by adding content of
wash buffer bottle provided with the kit to distilled
or deionized water to reach a final volume of 1
liter. The working wash buffer is stable for 2
weeks at 15-30°C. 25
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26. 3. Dispense 1 mL of Extraction Solution into Specimen
Extraction tube.
For Solid Stool Specimens:
i. Take out the cap of the Specimen Extraction Tube
ii. Randomly stab the specimen collection stick into the
stool specimen in at least 3 different sites to collect
approximately 30 mg of specimen (equivalent to 1/4 of a
pea). Do not scoop the stool specimen.
iii. Transfer into Specimen Extraction Tube.
For Liquid Stool Specimens:
i. Hold the Liquid Specimen Dropper vertically.
ii. Aspirate stool specimens and then dispense 2 drops
(approximately 50 μL) into the Specimen Extraction
Tube containing the Extraction Solution.
iii. Screw on and tighten the cap onto the Specimen
Extraction Tube.
iv. Shake the Specimen Extraction Tube vigorously to
mix the specimen and the Extraction Solution. 26
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27. 4. Leave A1 as Blank well.
5. Dispense 50 μL of Calibrator 1 in wells B1 and C1.
(Yellow Reagent)
6. Dispense 50 μL of Calibrator 2 in wells D1 and E1. (Blue
Reagent)
7. Dispense 50 μL of Calibrator 3 in wells F1 and G1. (Blue
Reagent)
8. Dispense 50 μL of Calibrator 4 in wells H1 and A2. (Blue
Reagent)
9. Hold the Specimen Extraction Tube upright and break off
the tip of the tube. Invert the Specimen Extraction Tube and
dispense 2 drops of the specimen Extraction Solution
(approx. 50 μL) to assigned wells starting at B2. (Yellow
Reagent)
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28. 10. Dispense 50 μL of Conjugate to each well except for
the Blank well. (Red Reagent)
11. Mix gently by swirling the micro-well plate on a flat
bench for 30 seconds.
12. Cover the micro-well plate with the Plate Sealer and
incubate at room temperature (15-30°C) in a room, a water
bath, or an incubator for 60 minutes ± 5 minutes.
13. Remove the Plate Sealer.
14. Wash each well 5 times with 350 μL of Working Wash
Buffer per well, and then remove the liquid.
15. Turn the micro-well plate upside down on absorbent
tissue for a few seconds. Ensure that all wells have been
completely washed and dried. Note: Improper washing may
cause false positive results.
16. Dispense 50 μL of Substrate A to each well. (Clear
Reagent) 28
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29. 17. Dispense 50 μL of Substrate B to each well. (Clear
Reagent) Then a blue color should develop in wells
containing Positive specimens.
18. Mix gently then cover micro-well plate with Plate Sealer
and incubate at room temperature (15-30°C) in a room, a
water bath, or an incubator for 10 minutes ± 1 minute.
19. Remove the Plate Sealer.
20. Dispense 50 μL of Stop Solution to each well. (Clear
Reagent) Then a yellow color should develop in wells
containing Positive specimens.
21. Read at 450/630-700 nm within 30 minutes.
Note: Micro-well plate can also be read at 450 nm, but it is
strongly recommended to read it at 630-700 nm for better
results.
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30. RESULTS INTERPRETATION
Minimum detectable concentration: 0.5ng/ml
Negative: < 15 ng/ml
Positive: > 20 ng/ml
Borderline: between 15-20 ng/ml, retest is recommended.
QUALITY CONTROL
 H. pylori Control containing both negative and positive
controls be run with each batch of samples to monitor
the procedure.
 QC intervals and limits should be adapted to each
laboratories individual requirements.
 Each laboratory should establish corrective measures if
values fall outside the limits.
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31. LIMITATIONS
 Diagnosis of an infectious disease should not be
established based on a single test result. Further testing,
including confirmatory testing, should be performed
before a specimen is considered positive.
 As with other sensitive immunoassays, a false positive
result may arise due to inadequate washing from the
initial test. The results may be affected due to procedural
or instrument error.
 A negative test result does not exclude the possibility of
exposure. Specimens containing precipitate may give
inconsistent test results.
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32. 2. Rapid Antigen test
INTENDED USE
 One Step H. pylori Antigen Test is an in vitro qualitative
immunochromatographic assay for the rapid detection of
Helicobacter pylori antigens in human stool specimen.
 The test results are intended to aid in the diagnosis of H.
pylori infection, to monitor the effectiveness of therapeutic
treatment and to confirm the eradication of H. pylori in
peptic ulcer patients.
 The test detects directly antigens in specimens for an
active infection.
 The test is simple and easy to perform and the test results
can be visually interpreted within 15 minutes
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33. PRINCIPLE OF THE TEST
 It is a sandwich solid phase immunochromatographic
assay.
 To perform the test, an aliquot of diluted stool sample is
added to the sample well of the test cassette.
 The sample flows through a label pad containing H.
pylori antibody coupled to red-colored colloidal gold.
 If the sample contains H. pylori antigens, the antigen will
bind to the antibody coated on the colloidal gold particles
to form antigen-antibody-gold complexes.
 These complexes move on the nitrocellulose membrane
by capillary action toward the test line region on which
H. pylori specific antibodies are immobilized.
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34.  As the complexes reach the test line, they will bind to the
antibody on the membrane in the form of a line.
 A second red control line will always appear in the result
window to indicate that the test has been correctly
performed and the test device functions properly.
 If H. pylori antigen is not present or lower than the
detection limit of the test, only the control line will be
visible.
 If the control line dose not developed, the test is invalid.
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35. WARNINGS AND PRECAUTIONS
 For in vitro diagnostic use.
 Wear protective glove while handling kit components
and test specimens.
 Patient specimens and inactivated Positive Control may
contain infectious agents and should be handled and
disposed of as potential biohazards.
 Do not use kit components beyond expiration date.
 Dispose all used materials in appropriate container.
Treat as potential biohazard.
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36. MATERIALS AND REAGENTS
 OneStep H. pylori Antigen RapiCard InstaTest.
 Each cassette contains a test strip with H. pylori specific
antibody on the test region of the membrane and colored
H. pylori antibody-gold conjugate pad.
 Sample bottle.
Each sample bottle contains 1 ml of stool specimen
collection buffer. Store at 4-30o C
 Specimen collection container
 Timer.
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37. STORAGE INSTRUCTION FOR
MATERIALS
 The expiration date is indicated on the package label.
 Store Sample Collection Tubes at 4-30° C.
 Test device can be stored at 4-30 ° C
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38. SPECIMEN COLLECTION AND STORAGE
 Stool specimens should be collected in containers that do
not contain media, preservatives, animal serum or
detergents as any of these additives may interfere with
the test quality.
 Specimens may be stored at 2-8° C for 3 days without
interfering with the assay performance.
 For long-term storage of specimens, -20° C or colder is
recommended.
 Repeated freezing and thawing of specimens is not
recommended and may cause erroneous results.
 Do not store specimens in self-defrosting freezers.
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39. REAGENT PREPARATION
Bring all reagents, including test device, to room
temperature (20-30° C) before use.
SPECIMEN PREPARATION
 Unscrew the sample bottle, use the attached applicator
stick attached on the cap to transfer small piece of stool
(5-6 mm in diameter; approximately 100 mg 200 mg/0.1-
0.2 g) into the sample bottle containing specimen
preparation buffer.
 Replace the stick in the bottle and tighten securely.
 Mix stool sample with the buffer thoroughly by shaking
the bottle for a few seconds.
Note: Watery or diarrhea specimens are inappropriate for
testing. 39
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40. ASSAY PROCEDURE
1. Bring all materials and specimens to room temperature
(8 30° C).
2. Remove the Cassette from the sealed foil pouch.
3. Hold the sample bottle upright with the tip point toward
the direction away from the test performer, snap off the
tip.
4. Hold the bottle in a vertical position over the sample
well of the Cassette, deliver 3 drops ( 120 -150 µL ) of
diluted stool sample to the sample well.
5. Read the result within 15 minutes.
A strong positive sample may show result earlier.
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41. INTERPRETATION
Positive result: A distinct pink colored band appears on
test line regions, in addition to a pink line on the control line
region.
Negative result: No line appears in the test line region. A
distinct pink line shows on the control line region.
Invalid: The control line next to the test line does not
become visible within 15 minutes after the addition of the
sample.
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42. QUALITY CONTROL
 The control band is an internal reagent and procedural
control. It will appear if the test has been performed
correctly and the reagents are reactive.
 Good Laboratory Practice recommends the daily use of
control materials to validate the reliability of the device.
 Control materials which is not provided with this test kit
may be commercially available.
LIMITATIONS
 The test is for qualitative detection of H. pylori antigen in
stool sample and dose not indicate the quantity of the
antigens
 The test is for in vitro diagnostic use only.
 The test result should be used only to evaluate with
patient with signs and symptoms of gastrointestinal 42
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43. 43
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44. 3. Helicobacter pylori IgG ELISA
Kit
INTENDED
Use The Helicobacter pylori IgG ELISA Kit is intended
for use in evaluating the serologic status to H. pylori
infection in patients with gastrointestinal symptoms.
PRINCIPLE OF THE ASSAY
Purified H. pylori antigen is coated on the surface of
micro-wells.
 Diluted patients serum is added to the wells, and the
H. pylori IgG- specific antibody, if present, binds to
the antigen.
 All unbound materials are washed away.
 Enzyme conjugate is added, which binds to the
antibody-antigen complex. 44
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45.  Excess enzyme conjugate is washed off and substrate
and chromogen are added.
 The enzyme conjugate catalytic reaction is stopped
at a specific time.
 The intensity of the color generated is proportional
to the amount of IgG-specific antibody in the sample.
 The results are read by a micro-well reader
compared in a parallel manner with calibrator and
controls.
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46. PRECAUTIONS FOR USE
o Consider all samples received for analysis potentially
positive for infectious agents including HIV and the hepatitis
B virus.
o Observe universal precautions.
o Wear gloves, lab coat, and safety glasses when handling all
human blood products and infectious viruses.
o Place disposable plastic, glass, paper, and gloves that
contact blood in a biohazard bag or discard pan to be
autoclaved.
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47. Reagent and standard materials
1. H. pylori antigen coated micro assay plate
2. Serum Diluent
3. Calibrator
4. Horseradish-peroxidase (HRP) Conjugate
5. Chromogen/Substrate Solution
6. Wash Buffer (20X concentrate)
7. Stop Solution
8. Deionized water
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48. REAGENT PREPARATION
 All reagents should be allowed to reach room
temperature (18-25°C) before use.
 Dilute 1 volume of Wash Buffer (20×) with 19
volumes of distilled water
SAMPLE PREPARATION
 Serum should be prepared from a whole blood
specimen obtained by acceptable medical techniques.
This kit is for use with serum samples without
additives only.
 Specimens may be refrigerated at 2-8°C for up to 7
days or frozen for up to 6 months. Avoid repetitive
freezing and thawing of serum sample.
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49. ASSAY PROCEDURE
1. Secure the desired number of coated wells in the
holder.
2. Prepare 1:40 dilution of test samples, negative
control, positive control, and calibrator by adding 5
µl of the sample to 200 µl of sample diluent. Mix
well.
3. Dispense 100 µl of diluted sera, calibrator, and
controls into the appropriate wells. For the reagent
blank, dispense 100 µl sample diluent in 1A well
position. Tap the holder to remove air bubbles
from the liquid and mix well for 10 seconds.
4. Incubate at room temperature for 30 minutes
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50. 5. At the end of the incubation period, remove liquid from
all wells. Rinse and flick the microtiter wells 4 times with
diluted wash buffer (1×) and then one time with distilled
water. (Please do not use tap water.)
6. Dispense 100 µl of enzyme conjugate to each well. Mix
gently for 10 seconds.
7. Incubate at room temperature for 30 minutes.
8. Remove enzyme conjugate from all wells. Rinse and
flick the microtiter wells 4 times with diluted wash buffer
(1×) and then one time with distilled water.
9. Add 100 µl of TMB Reagent to each well. Mix gently for
10 seconds.
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51. 10. Incubate at room temperature for 20 minutes.
11. Add 100 µl of Stop Solution to each well including the 2
blanks.
12. Mix gently for 30 seconds. It is important to make sure
that all the blue color changes to yellow color
completely.
13. Read the optical density at 450 nm with a microtiter
plate reader.
Note: The wash procedure is critical. Insufficient washing
will result in improper color development.
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52. INTERPRETATION
Negative: H. pylori IgG EIA Index less than 0.90 is
seronegative for IgG antibody to H. pylori. The serum
sample may have been taken too early.
Equivocal: H. pylori IgG EIA Index between 0.91-0.99
is equivocal. Retest In a parallel fashion with a new
serum Sample drawn 3 weeks later.
Positive: H. pylori IgG EIA Index of 1.00 or greater is
seropositive.
QUALITY CONTROL
The test run may be considered valid provided the
following criteria are met:
 The O.D. value of the reagent blank against air from
a micro-well reader should be less than 0.250.
 If the O.D. value of the Cut-off Calibrator is lower
than 0.250, the test is not valid and must be
repeated.
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53. 4.Rapid detection of Antibodies
to H. pylori
INTENDED USE
The H. Pylori Rapid Test is a chromatographic immunoassay
(CIA) for the rapid determination of antibodies to H. pylori in
serum and/or whole blood specimens.
PRINCIPLE OF THE TEST
 The CD H. pylori Antibody Rapid Test is a lateral flow
immunochromatographic screening test.
 H. pylori specific antigens are pre-coated onto membrane
as a capture reagent on the test band region.
 During the assay the whole blood or serum specimen is
first allowed to react with H. pylori specific antigen-gold
conjugate complexes.
 The mixture then moves laterally on the membrane to the
test region which is coated with immobilized antibodies to
H. pylori. 53
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54.  If H. pylori antibodies are present in the specimen, a color
band is formed on the test (T) region.
 Absence of the colored band in the test region indicates a
negative result.
 To serve as a procedural control, a colored band in the
control (C) region will always appear regardless the
presence of H. pylori antibodies in the whole blood and/or
serum specimen
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55. PRECAUTIONS
 Do not use after expiration date printed on the outside of
the foil pouch.
 Test device should remain sealed until ready for use.
 There should be no smoking or eating where antigen-
containing material is handled.
 Decontaminate area by disposing samples and all
potentially contaminated materials as if they contained
infectious agents.
 Wear disposable gloves while handling samples.
 Wash hands thoroughly afterwards. 55
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56. REAGENTS AND MATERIALS PROVIDED
 A test device is packed in a protective, sealed foil pouch.
 Test running buffer ready for use.
STORAGE
 The CD H. Pylori Rapid Test is lo be stored refrigerated
or at room temperature (2-28°C) in the sealed pouch for
the duration of the product shelf life.
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57. SPECIMEN COLLECTION AND
PREPARATION
Whole blood specimen collection:
1.Collect fresh blood specimens just prior to using the assay.
2.One drop of whole blood is enough for the assay.
Serum specimen collection:
1.Collect and centrifuge blood specimen following standard
laboratory procedures.
2.Remove serum as soon as possible to avoid hemolysis.
Lipemic, icteric, or hemolyzed specimens may give
inconsistent test results.
Specimens containing a precipitate should be clarified prior
to testing.
3.If specimens cannot be tested immediately, they should be
stored refrigerated at 2-8°C for no longer than 3 days. 57
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58. ASSAY PROCEDURE
1. Remove the device from the protective pouch and label
the device with specimen identification.
2. Add one drop (50 µl) of whole blood or one drop (25 µl)
of serum to the Sample Well (for Card) or the Sample
Pad (for Dipstick). Then add 3 drops (150 µl) of test
running buffer lo the Sample Well or Sample Pad.
3. Read the result within 10 minutes.
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59. INTERPRETATION OF
RESULTS
Positive: Presence of two colored
bands, one in the control region
(C) and another in the test region
(T), indicates presence of
antibodies to H. pylori in specimen.
Negative: Presence of a colored
band in the control region (C) with
no band in the test region (T)
indicates absence of antibodies to
H. pylori in the specimen.
Invalid: If after 10 minutes no band
is visible, or if a band appears in
the test region (T) only, the result is
invalid.
If invalid, the assay should be
repeated using a new lest kit.
Note: Do not interpret results after
15 minutes 59
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60. LIMITATIONS
 The H. Pylori Rapid Test is not reusable.
 The test works only if the instructions are followed
precisely.
 Do not use the test after the expiration date shown on
the package or if the moisture absorbent pack is wet.
 Serum from samples where C. jejuni is present may
have a low cross-reactivity with this test.
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