Helicobacter pylori and IHC

(immunohistochemistry)

image from : en.wikipedia.org

Lauri Zaker

Bacteria and viruses predate us by one billion years

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.

H. pylori :

gram negativelophotrichous (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 growlike neutral pH environment

http://bioweb.uwlax.edu/

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

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 stomachFrom 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

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.

Ways to Test for H.pylori infectionUrease Breath Test

Stool Sample (blotting)

Endoscopy/ Biopsy w/FFPE and special stainsor

IHC /biopsy w/ antibodies and conjugated epitope markers on FFPE or

w/ epitope markers onELISA -type blotting strips (enzyme-linked immunosorbent assay)

Double Antigen Chromatographic Lateral Flow ImmunoassayThe 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.

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% respectivelyFor 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."

Comparison of IHC, toluidine blue, Giemsa and H&EJanuary 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).

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.

A vaccine is on its way

Image: Coulourbox

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.

ReferencesInfections in Cancer by Brock, T.G.

Ulcers Discovered in Mummies by Live Science Staff http://www.livescience.com/2696-ulcers-discovered-mummies.html

microbeWikiComparison 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): 136-40A 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.2007.02.031Source: PubMed Towards the first Helicobacter pylori vaccine? Technologist Online Nov.6,2014http://www.technologist.eu/towards-the-first-helicobacter-pylori-vaccine/

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]