1. Helicobacter pylori
and IHC(immunohistochemistry)
image from : en.wikipedia.org
Lauri Zaker

2. Bacteria and viruses predate us by one billion years

3. Helicobacter pylori has been coexisting within humans for at least 60,000 years.
Image: newscientist.com
Human migration pattern. Greatest number of strains in Africa.
Second mass migration out of Africa was ~ 60,000 years ago.
Also, H.pylori was found in 3,000 year old mummies in Mexico and had caused gastritis.

4. H. pylori :
gram negative
lophotrichous (two or more flagella on one end
only)
spirillae (spiral shaped)
(can change shape due to circumstances)
microaerophilic (need little air)
fastidious (need certain nutrients to grow,i.e.
nickel , iron , CO2, blood--(alternatively, egg
yolk or b cyclodextrin))
take 4 or more days to grow
like neutral pH environment
http://bioweb.uwlax.edu/

5. Helicobacter pylori: Physiology and Genetics.Chapter 4Basic
Bacteriology and Culture
Lief Percival Andersen and Torkel Wadström.
H. pylori transforms into coccoid forms
under certain conditions such as
nutrient starvation and media
containing growth inhibitors (bismuth,
proton pump inhibitor, or certain
antibiotics). These coccoid forms have
been reported to survive for several
years in river water and have been
proposed by some to be an important
factor for transmission, by fecal
excretion, and for therapy failure.
Image:
http://www.phmd.pl/fulltxthtml.php?ICID=1044005

6. H. pylori typically reside in the antrum of
the stomach, whereas the greatest acid
secretion, ulceration, and tumor formation
occur in the body, or corpus, of the stomach
From Infections in Cancer by Brock, T.G.
H. pylori are a slow growing
organisms that can cause peptic
ulcers and gastritis that can lead to
gastric cancer and gastric MALT
(mucosa-associated lymphoid
tissue) lymphoma.
from: microbeWiki

7. H.pylori has many tricks up its sleeve, growing
a pilus and injecting host cells with toxins and
using its effector proteins to hijack the host
cell’s machinery to send out cytokines to lure
more immune cells to come and it kills
whoever comes , through apoptosis. It is now
known to be able to stop helper T cell
proliferation also.

8. Ways to Test for H.pylori infection
Urease Breath Test
Stool Sample (blotting)
Endoscopy/ Biopsy w/FFPE and special stains
or
IHC /biopsy w/ antibodies and conjugated epitope markers on FFPE
or
w/ epitope markers on
ELISA -type blotting strips (enzyme-linked immunosorbent assay)

9. Double Antigen Chromatographic Lateral Flow Immunoassay
The test strip in the device includes:
1) a rose pink-colored conjugate pad containing colloidal gold coupled with H. pylori antigens
2) nitrocellulose membrane containing a test line (T line) and a control line (C line).
The T line is coated with H. pylori antigens, and the C line is coated with goat anti-H. pylori antibody.
The antigens used in this device are from H. pylori cell lysate. When IgG antibodies specific to H. pylori are present in the
specimen, the T line will become a rose pink-colored band. If antibodies to H. pylori are not present or are present below the
detectable level, no T line will develop.
The C line should always appear as a rose pink-colored band regardless of the presence of antibodies to H. pylori.
The C line serves as an internal qualitative control of the test system to indicate that an adequate volume of specimen has
been applied and the flow occurred.
Detection: + or - of infection.
Doesn’t necessarily show specificity
Doesn’t show quantification.

10. From a small study in India, 2011:
Comparison of enzyme immunoassays detecting Helicobacter pylori
specific IgG in serum and saliva with endoscopic and biopsy findings in
patients with dyspepsia.
biopsies. 55 patients Their findings were: sensitivity: 90.5% and 95% respectively
specificity:84.5% and 70% respectively
For diagnosing H.pylori associated duodenal ulcer and gastritis:
“we are of the opinion that immunoassays detecting HPspecifi c IgG antibodies in saliva are
simple and practical in clinical settings and are concordant with invasive tests. Furthermore, as
suggested by some authors, such tests, can be considered as good as the gold standard biopsy
test in diagnosing HP infection and screening of dyspeptic patients.”
They said :" The performance characteristics of the enzyme immunoassays currently used
in detecting salivary or serum IgG can be further refined by introducing IgG capture
immunoassays utilizing highly specific enzyme labeled HP (H.pylori) antigens."

11. Comparison of IHC, toluidine blue, Giemsa and H&E
January 2013, a small study was done in Iran to compare H&E, Giemsa and
toluidine blue staining with immunohistochemistry for the detection of H. pylori in
patients who were referred for upper endoscopy with or without medication
history.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600970/
. (A) The obvious identification of cluster
of modified coccoid forms of Helicobacter
pylori by immunohistochemistry; (B)
coccoid H. pylori on Giemsa stained section
(C) IHC stain forH. pylori shows a small
area with organisms inside the epithelial
cells. Inset shows individual H. pylori with
characteristic elongated, slightly spiral S
shaped; (D) Giemsa stained biopsy of the
same sample which does not show any H.
pylori organism.
(A) Clusters of immunolabeled
organisms at the surface of pyloric
mucosa of a patient infected with
Helicobacter pylori,
immunohistochemical staining for
H. pylori (primary antibody:
Bo471 DAKO, Denmark),
hematoxylin counterstain, positive
control reaction; (B) negative
control reaction.
Histochemical staining. Bacteria are
visualized (A) in the lumen of antral gastric
glands on H&E stain (arrows), (B) more easily
in modified Giemsa stained section (arrows)
and (C) in the lumen of antral gastric glands
on toluidine blue stain (arrow).

12. Their conclusions, which are in line with many other papers I read:
In the discussion of their results, they found that :
- H&E had the sensitivity of 41.86% and the specificity of 100% : which they found inadequate for detection of
H.pylori.
- Giemsa method exhibited a sensitivity of 53.74% and specificity of 95.24%, which surprised them at being so
low.
- Toluidine blue sensitivity and specificity was 76.74% and 100%, respectively.They found that this method is
cheap and easy to use and produces more reliable results than H&E/modified Giemsa method.The major
disadvantage of this method is that there is little contrast between organisms and tissues.
Immunohistochemistry is a reliable technique for detection of H. pylori. Coccoid forms of the
organisms, which may not be amenable to other staining methods, were seen easily on
immunostained sections. Also, H. pylori antigen in the lamina propria and beneath the surface
epithelia is detectable by IHC, while it can be hardly detectable by histochemical stains. On the
other hand, the method is fairly expensive, especially because a negative control needs to be
used with every slide. Therefore, it is not clearly practical and economical to perform H. pylori
immunohistochemistry on every gastric biopsy specimen. However, in cases with inactive
gastritis or low degree of inflammation, and cases with chronic active gastritis with negative
histological result, IHC stain should be superior to histochemical stains in detection of H. pylori.

13. A vaccine is on its way
Image: Coulourbox

14. Mark Gerhard , professor of medical microbiology and immunology in Germany,
started a company which plans to start a Phase 1 human trial in 2016 .
Based on findings in 2007, his group found that H.pylori has a way to stop T
helper cell proliferation in the G1 phase of the host organism.They have come
up with a way to prevent H.pylori from doing that.
The hardest part of making it is that its not a prophylactic vaccine , but a
therapeutic vaccine.
Different than a vaccine to prevent future infection, a therapeutic vaccine enlists
the body's immune system to fight an existing infection.

15. References
Infections in Cancer by Brock, T.G.
Ulcers Discovered in Mummies by Live Science Staff
http://www.livescience.com/2696-ulcers-discovered-mummies.html
microbeWiki
Comparison of enzyme immunoassays detecting Helicobacter pylori specific IgG in serum and saliva with endoscopic and
biopsy findings in patients with dyspepsia Indian Journal of Medical Microbiology, (2011) 29(2): 136-40
A El-Mekki, *A Kumar, B Alknawy, O Al-Ammari, R Moosa, S Quli, M Ahmed
Inhibition of T-cell proliferation by Helicobacter pylori gamma-glutamyl transpeptidase.Gerhard M., Rad,R. et al.
Department of Medicine II, Technical University of Munich, Munich, Germany.
Gastroenterology (Impact Factor: 13.93). 06/2007; 132(5):1820-33. DOI: 10.1053/j.gastro.2007.02.031
Source: PubMed
Towards the first Helicobacter pylori vaccine? Technologist Online
Nov.6,2014http://www.technologist.eu/towards-the-first-helicobacter-pylori-vaccine/

16. References (continued)
Tajalli, Raziye et al. “The Immunohistochemistry and Toluidine Blue Roles forHelicobacter Pylori
Detection in Patients with Gastritis.” Iranian Biomedical Journal 17.1 (2013): 36–41. PMC. Web. 19
Feb. 2015.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600970/
Helicobacter pylori: Physiology and Genetics. Chapter 4 Basic Bacteriology and Culture Lief Percival Andersen and Torkel
Wadström.
Eaton K. A., Catrenich C. E., Makin K. M., Krakowka S. Virulence of coccoid and bacillary forms ofHelicobacter pylori in gnotobiotic piglets. J. Infect. Dis.
1995;171:459–462. [PubMed]
Bode G., Malfertheiner P., Ströhle A., Mauch F., Nilius M., Ditschuneit H. Polymorphismus bei Helicobacter pylori—Schlüsselfunktion für
Infektionsrezidive? Med. Klin. 1992;87:179–184. [PubMed]
Catrenich C. E., Makin K. M. Characterization of the morphologic conversion of Helicobacter pylori from bacillar to coccoid forms. Scand. J. astroenterol.
1991;26(Suppl. 181):58–64. [PubMed]