Table Of ContentIntroduction: Medical Significance of H. pylori
Martin J. Blaser
Until the discovery of Helicobacterpylori in 1982, the normal human stom-
ach was generally considered to be sterile, or transiently populated by oropha-
ryngeal bacteria carried there by peristalsis. However, we now know that from
one-third to one-half of the human population carries H pylori, and that once
infected, most persons remain infected for decades, if not for life (I).
H. pylori infection is almost invariably associated with gastric inflammation
and induces the most common inflammatory lesion, known as chronic superfi-
cial gastritis (2). This lesion is so common, especially among the elderly, that it
was thought to represent the aging stomach, but we now know that eradication
of H. pylon from the stomach results in clearance of this pathology (3). How-
ever, based on present knowledge, those persons with H. pylori-induced
chronic superficial gastritis do not have any symptoms. Until the discovery of
H pyZori, chronic superficial gastritis was essentially a medical curiosity, appre-
ciated only by pathologists who could not agree about the nosology of gastritis.
H. pylori Association with Gastric Diseases
Now it is clear that H. py2ori infection is highly associated with a number of
the most important diseases of the upper gastrointestinal tract. These include
duodenal and gastric ulcers, gastric adenocarcinoma, and non-Hodgkin’s
lymphomas of the stomach (Table 1). Although H pylori infection IS not
present in all cases of these disorders (Fig. l), there IS a substantial body of
evidence that it is involved in the causation of each of these (4-12). Conversely,
there are other diseases of the upper gastrointestinal tract with which H pylori
infection is not associated, including reflux esophagitis, Barrett’s esophagus,
bile reflux gastritis, pernicious anemia, and the gastropathy associated with
use of nonsteroidal anti-inflammatory drugs (NSAIDS). Thus, H. pylori is
From Methods 1r-1M olecular Mehne, Helicobacter pylon Protocols
Ed&d by. C. L. Clayton and H L T. Mobley Humana Press Inc , Totowa, NJ
I
2 Blaser
Table 1
Association of H. py/ori Infection with Important Inflammatory
and Neoplastic Conditions of the Upper Gastrointestinal Tract
Estimated
Condition odds ratio Refs.
Peptrcu lcer disease 4-12 43
Adenocarcinomao f the stomach 8 6-9
Non-Hodgkin’s lymphoma of the stomach 6 10,rr
Barrett’s esophagus 1 12
Uninfected
Fig. 1. H pylori infection, ulcer disease,a trophic gastritis, and gastric neoplasra.
Associationo f H. pylon infection and medical condttronso f the upper gastrointestinal
tract as shown m a Venn diagram. DU, duodenal ulcer; GU, gastric ulcer; CAG,
chronic atrophic gastntls; GC, gastrx cancer (adenocarcinoma of the stomach), L,
Non-Hodgkin’s lymphoma of the stomach.
related to risk of certain diseases of the upper gastrointestinal tract, but 1sn ot
nonspecifically associated with them all.
Not All H. pylorblnfected Persons Become Ill
A seeming paradox is that most infected persons never develop any conse-
quences of colonization with H pyZori during the decades-long course of the
infection; this is not unusual for other host-adapted agents of chronic mfec-
tious diseases. For example, Mycobacterium tuberculosis infects about
one-third of the world’s population, as well, yet rt has become clear that less
than 10% of normal hosts will develop any clinical consequences (most com-
monly, pulmonary tuberculosis) of the infection. The other 90% have no clim-
Medical Significance of H. pylori 3
H. pylon Mection
Weeks-Months
0
Fig. 2. Natural history of H pylorr infection. Within months after H pylon is
acquired, infected persons develop chronic superfictal gastrrtis. This persists, essen-
tially for life, in most infected persons. However, one subgroup of infected persons
develops peptic ulceration, another develops chrome (multifocal) atrophic gastritis,
which may lead to adenocarcinoma of the stomach, and a third group develops
lymphoproliferative disorders, such as MALT-type lymphomas. Reprinted with per-
mission from ref. 23.
tally apparent consequences during their lifetime. Examining the clinical data
another way, we find that among persons with chronic destructive pulmonary
inflammation, with cavities, the vast majority are caused by A4. tuberculosis,
and a few result from other disorders, such as Wegener’s granulomatosis and
chronic fungal infections. This paradigm nearly parallels that known for
H. pylori and peptic ulceration. Although <lo% of H. pylon-infected persons
develop ulceration during the course of their infection, H. pylori is responsible
for >95% of those with idiopathic (non-NSAID or Zollinger Ellison-induced)
ulcers (23).
Similarly, even in parts of the world where gastric cancer is hyperendemic,
less than 5% of H. pylori-infected persons develop these neoplasms. How-
ever, in parallel with the ulcer data, attributable risk analysis suggests that
H. pylori is responsible for 60430% of these tumors (9).
Natural History of H. pylori-Infection
From studies conducted by hundreds of investigators all over the world, it
has become clear that H pylori infection has a natural htstory (Fig. 2). Within
4 Blaser
weeks after the organism is acquired, chronic superficial gastritis develops
(14,15). In most persons, this lesion persists, without any consequences, for
the duration of the infection (2). One subgroup of patients develops peptic
ulcer disease, another develops a variety of lymphoproliferative disorders,
and in others, chronic superficial gastritis progresses to atrophic gastritis.
The latter lesion is important because it is the major recognized risk factor
for the development of gastric cancer. The development of cancer probably
takes 3-6 decades of infection to manifest. An important aspect in under-
standing H. pylori infection is to consider why this diversity of outcomes
develops. The determinants for specific consequences of infection may be
divided into four categories. First, there may be differences among H. pylori
strains m their virulence potential. Second, there may be important differ-
ences among infected hosts in their genetic characteristics. Third, exposure
of hosts to environmental cofactors (such as smoking or particular dietary
substances) differs and may relate to outcome of H. pylori infection. Finally,
as with other pathogenic agents infecting humans, the age at which H. pylorz
is acquired may affect outcome. In fact, there now is evidence for each of
these possibilities (1619). The determinants of outcome are multifactorial
and complex.
Management of H. py/ori Infection
Over the last decade, important advances have permitted the ready and
accurate diagnosis of H pylori mfection (20). Similarly, antimicrobial therapy
has now improved to the point that safe and well-tolerated relatively short term
(7-10 d) regimens are more than 90% effective (22,22). It now is clear that
H. pylori-infected persons who develop peptic ulceration or (MALT) lymphomas
should be treated, and perhaps an argument can be made for those with family
or exposure histories that point to high risk of developing gastric cancer. How-
ever, at this time it is premature to consider eradicating H. pylori infection in
everyone. Defimtion of risk factors for the development of specific diseases
will facilitate this quest. Similarly, investigators also should focus on whether
this well-adapted and persistent bacterium may have any beneficial effects for
humans. If such properties are discovered, or if there are commensal H pylori,
there may be individuals in whom infection should be maintained,
Conclusions
In the 14 yr since the discovery of H. pylori, there has been remarkable
scientific progress. We have learned about the role of these organisms m
important human diseases, methods of diagnosis are readily available, and
effective treatments have been found. An improved understanding of the patho-
genesis of infection will facilitate further medical progress.
Medical Significance of H. pylori 5
References
1. Pounder, R. E. and Ng, D. (1995) The prevalence of Helicobacterpylori infection
m different countries. Aliment Pharmacol. Ther 9, S33-S39.
2. Dooley, C P., Fitzgibbons, P. L., Cohen, H , Appleman, M. D., Perez-Perez, G I.,
and Blaser, M J. (1989) Prevalence of Helicobacter pylori infection and histo-
logic gastritis in asymptomatic persons. N. Engl. J Med 321, 1562-l 566
3. Blaser, M J. (1990) Helicobacterpylori and the pathogenesis of gastroduodenal
inflammation. J. Infect. Dzs. 161,626-633
4. Stpponen, P., Helske, T., Jarvmen, P., Hyvarinen, H., Seppala, K., and Smrala, M.
(1994) Fall in the prevalence of chronic gastritis over 15 years: analysts of outpa-
tient series in Finland from 1977, 1985, and 1992. Gut 35, 1167-l 171.
5. Nomura, A., Stemmermann, G. N., Chyou, P., Perez-Perez, G. I , and Blaser, M.
J. (1994) Helicobacterpylori infection and the risk for duodenal and gastric ulcer-
ation Ann Intern Med. 120,977-981.
6. Nomura, A., Stemmermann, G. N., Chyou, P., Kato, I., Perez-Perez, G I , and
Blaser, M. J. (199 1) Helicobacterpylori infection and gastric carcmoma m a popu-
lation of Japanese-Americans in Hawaii N. Engl J, Med 325, 1132-l 136
7 Parsonnet, J., Friedman, G. D., Vandersteen, D. P , et al. (1991) Helzcobacter
pylon infection and the risk of gastric carcinoma. N Engl. J Med 325, 1127-l 13 1
8. Forman, D., Newell, D. G., Fullerton, F., Yarnell, J. W., Stacey, A. R., Wald, N.,
and Sitas, F. (1991) Association between infection with Helicobacterpylori and
risk of gastric cancer: evidence from a prospective investigation. BMJ 302,
1302-1305.
9. Forman, D. (1995) The prevalence ofHelicobacterpylorz infection in gastric can-
cer. Aliment. Pharmacol Ther 9, S71-S76.
10. Parsonnet, J., Hansen, S., Rodriguez, L., Gelb, A. B , Warnke, R. A., Jellum, E.,
Orentretch, N., Vogelman, J. H., and Friedman, G. D. (1994) Helxobacterpylori
infection and gastric lymphoma. N Engl J Med 330, 1267-1271.
11. Wotherspoon, A. C., Doglioni, C., Diss, T. C., et al. (1993) Regression of primary
low-grade B-cell lymphoma of mucosa-associated lymphoid tissue type after
eradication of Helicobacterpylori. Lancet 342,575-577.
12. Blaser, M. J., Perez-Perez, G. I., Lindenbaum, J., Schneidman, D., VanDeventer,
G., Marin-Sorenson, M., and Weinstein, W. M. (1991) Association of mfection
due to Helwobacter pylori with specific upper gastrointestinal pathology. Rev
Injkt. Dis 13, S704-S708.
13. Kuipers, E. J., Thijs, J. C., and Festen, H. P. M. (1995) The prevalence of
Helicobacterpylori in peptic ulcer disease. Aliment. Pharmacol Ther 9, S59-S69.
14. Morris, A. and Nicholson, G. (1987) Ingestion of Campylobacterpylondrs causes
gastritis and raised fasting gastric pH. Am. J, Gastroenterol. 82, 192-l 99.
15. Morris, A. J., Ali, M. R , Nicholson, G. I., Perez-Perez, G I., and Blaser, M. J.
(199 1) Long term follow-up of voluntary ingestion of Helicobacter pylon. Ann.
Intern.Med 114,662,663.
16 Figura, N., Guglielmetti, P., Rossolini, A., Barberi, A , Cusi, G., Musmanno, R.
A., Russt, M., and Quarantas, S. (1989) Cytotoxin production by Campylobacter
6 Blaser
pylori strains isolated from patients with peptic ulcers and from patients with
chronic gastritis only. J, Clin Mlcroblol. 27,225,226.
17 Cover, T. L., Dooley, C. P., and Blaser, M. J. (1990) Characterization of and
human serologrc response to proteins m Helrcobacter pylorl broth culture super-
natants with vacuolizmg cytotoxm activity. Infect. Immun. 58,603-6 10.
18. Malaty, H. M., Engstrand, L., Pedersen, N L., and Graham, D. Y (1994)
Helicobacter pylorl infection: genetic and environmental influences A study of
twins. Ann Intern Med 120,982-986.
19. Blaser, M. J., Perez-Perez, G. I., Kleanthous, H., Cover, T. L., Peek, R. M , Chyou,
P. H., Stemmerman, G. N., and Nomura, A. (1995) Infection with Hekobacter
pylon strains possessmg cagA associated with an increased risk of developing
adenocarcinoma of the stomach. CancerRes. 55,2 11 l-2 115.
20. Feldman, R. A. and Evans, S. J. W (1995) Accuracy of diagnostic methods used
for epidemrologmal studies of Helrcobacterpylorl. Aliment Pharmacol. Ther. 9,
S21S31.
21. de Boer, W., Driessen, W., Jansz, A., and Tytgat, G (1995) Effect of acid sup-
pression on efficacy of treatment for Hellcobacter pylorz infection. Lancet 345,
817-820.
22. Lind, T., vanzanten, S. J. O., Unge, P., et al. (1995) The Mach I study: optimal
one-week treatment for H pylorl defined? Gut 37, A4.
23. Blaser, M. J. and Parsonnet, J (1994) Parasitism by the “slow” bacterium
Helicobacter pylon leads to altered gastric homeostasis and neoplasia. J. Clan
Invest 94,443
Detection of H. pyhi Infection
by Biopsy Urease, Histology, and Culture
Stewart Goodwin
1. Introduction
Detection of Helicobacter pylori in specimens obtained by endoscopy
requires the gastroenterologist to select suitable patients for endoscopy, and to
take an adequate number of biopsy specimens; there must also be correct clean-
ing of the biopsy forceps.
7.7. Selection of Patients for Endoscopy
Any antibiotic active against H. pylori will cause a reduction in the num-
bers of bacteria in the stomach. If the patient has been treated with such an
antibiotic or has ingested an over-the-counter preparation containing bismuth
such as Pepto-Bismol, in the preceding 4 wk before the endoscopy, then
endoscopy must be delayed until 4 wk after the last antibiotic treatment (1).
Also, if the patient has received a drug that reduces the acid in the stomach
and raises the pH, this will affect the area of the stomach to be biopsied.
Hz-receptor antagonists (ranitidine and cimetidine) raise the gastric pH, but
proton pump inhibitors, such as omeprazole and lanzoprazole, raise the
gastric pH to a higher level. When the gastric pH has been raised, H. pylon is
not so common in the antrum of the stomach, but is found in the corpus more
frequently than in patients who have not received such drugs (2); m such
patients, at least one biopsy specimen must be taken from the corpus and
the antrum.
Various chemical agents used during the endoscopy procedure can be anti-
bacterial to H. pylori. Benzocaine has a minimum inhibitory concentration for
From* Methods /n Molecular Me&he, Hellcobacter pylorr Protocols
Edlted by* C. L Clayton and H. L T. Mobley Humana Press Inc., Totowa, NJ
7
8 Goodwin
H. pylori of 0.14-0.7 mg/L, and simethicone is also inhibitory. Lidocame is
not inhibitory to H. py2ori.
1.2. Cleaning of Endoscopy Forceps
An efficient cleaning and disinfecting method for the biopsy forceps must
always be employed between each patient. If disinfection is inadequate, then
a subsequent patient(s) may become infected with H. pylon’ from a previous
patient. However, rarely will a positive culture be obtained from such
inadequately disinfected forceps. Another danger is that a strong disinfec-
tant, such as glutaraldehyde, may remain on the biopsy forceps and kill the
bacteria, rendering the culture falsely negative. Most modern endoscopes are
purchased with a machine that fully disinfects and cleans the instrument
between patients.
1.3. The Number of Biopsy Specimens
to be Obtained and Their Sites
The number of specimens to be taken is determined first by the range of
tests for H. pylori that will be requested. It is recommended that one specimen
be taken from the antrum for the rapid biopsy urease test. Histology is always
performed on one or more specimens, but some physicians avoid specimens
for microbiological processing because these are an extra expense. If several
biopsies are taken for histology and one specimen for the biopsy urease test,
detection of H. pylori may not need a specimen for microbiological process-
ing. However, an important opportunity with endoscopy is to determine the
antibiotic sensitivity of the isolate to guide correct antibiotic treatment. Because
H. pylori is not evenly distributed around the stomach surface, but has a patchy
distribution, the more biopsy specimens that are taken, the less likely it is that
the presence of H. pylori will be missed, and these extra specimens can be
processed microbiologically. At least one specimen for histology should be
taken from the antrum and one from the corpus. At least one specimen from
the antrum should be sent for microbiological processing and, to be sure of
detecting the organism, probably two specimens are required, so that one can
be processed for microscopy and the other for culture. Second, if the patient
has received a proton pump inhibitor, at least one biopsy specimen must be
taken from the corpus (2). All specimens should be kept at 4OC before and after
transport to the laboratory.
Detection of H. pylori involves a biopsy urease test, a histological process
that ensures detection of spiral bacteria, an efficient transport medium for
microbiology, correct microbiological microscopy and inoculum preparation
for culture on solid media with the correct incubation atmosphere, and length
of incubation. These are described in the followmg sections.
Detection by Biopsy, Histology, Culture 9
2. Materials
2.1. Tests in the Endoscopy Unit
2.1.1. Biopsy Urease Tests.
These testsd etect the presence of preformed urease produced by H pylon in
the biopsy specimen; growth of the organisms is not required. The urease of H.
pylori hydrolyzes urea in the medium, with the production of ammonium ions,
which raise the pH and change the color of the indicator.
2.1.1 .~.COMMERCIAL CL0 TEST
Delta-West Ltd., Bentley, Western Australia. This 2% urea unbuffered gel
preparation has proved its stability and reliability, and has been found to be
very accurate, when read after 3 h, with a sensitivity of 90% and a specificity
of 100% (3). However, a minimal amount of 10,000 cm/ml is necessary to
obtain positivity, and the sensitivity of all urease tests drops to 50% m patients
after antimicrobial treatment (4).
2.1 1 2. LIQUID UREA MEDIUM
This can be prepared in the microbiology laboratory and is usually cheaper
than the CL0 test. The concentration of urea has been varied by many workers,
but a medium with 6% urea is probably the most rapid and reliable. In theory,
higher concentrations may result in inhibition of the urease of H. pylori. The
ingredients for the liquid urea medium are: 60 g/L urea; 0.012 g/L phenol red;
2 g/L KH,PO,; 1 g/L peptone; 5 g/L NaCl; 10 g/L glucose, in distilled water.
Three-milliliter aliquots are dispensed into small containers with screw caps.
This solution, without buffer, produces the most rapid result, but is unstable
and should be made up fresh at least once a week.
2.1.2. Simple Carboifuchsin Staining
Carbolfuchsin solution prepared as follows: 0.4 g basic fuchsin, 2 g phenol
crystals, 4 mL absolute alcohol (made up to 100 mL in distilled water); abso-
lute alcohol, glass slides.
2.2. Histology (see Note I)
2.2.1. Imprint Cytology
Requirements: a glass microscopy slide, sterile needle, absolute alcohol, 2%
Giemsa solution (Giemsa concentrate, Difco, Detroit, MI) in distilled water.
2.2.2. Modified Giemsa Method
Requirements: 2% (v/v) Giemsa (see Section 2.2.1.) solution m distilled
water, absolute alcohol.
10 Goodwin
2.2.3. Simple Carbolfuchsin Staining
Requirements: 4% (v/v) neutral formaldehyde, distilled water, and carbol-
fuchsin solution prepared as in Section 2.1.2.
2.2.4. lmmunohistochemical Staining
Requirements: Antibodies MABOOl and MAB002 (Bioproducts for Science,
Indianapolis, IN).
2.3. Microbiological Processing of Specimens
2.3.1. Transport Media to the Laboratory (see Note 1)
1. H. pylon rapidly losesv iability at room temperaturei n 0.9% (w/v) NaCl, so this
should not be used unless the specimen can be received m the laboratory within 1 h.
It should be dispensed in 0 5-mL ahquots.
2 Brucella broth (Oxoid CM169 [Basmgstoke, UK]; BBL 11088 [Becton
Dickinson, Cockeysville, MD]; Difco 0495). These should be made up followmg
the mstructions of the maker, with the addition of 0 5% (w/v) bovine serum albu-
min. Three-milhllter aliquots should be dispensed into small containers with
screw caps
2.3.2. Preparation of lnoculum for Solid Media
Requirements: sterile forceps, normal saline, a ground-glass grinder or an
electric tissue grinder (Potter-Elvehjem homogenizer [Citenco, Boreham
Wood, Hertsfordshire, UK]) or a mechanical Teflon homogenizer, glutaralde-
hyde or alcohol (to clean the blades and parts of the grinder), and carbolfuchsin
solution (see Section 2.2.3.).
2.3.3. Phase Contrast Microscopy
A phase contrast microscope is required.
2.3.4. Culture on Solid Media (see Note 3)
1. Most authorities recommend maculation of one selective medium with antibiot-
ics, and also a medium without antibiotics. Freshly prepared moist plates are
essential to obtain the maximal isolation rate of H pylori, although if the fresh
plates are wrapped immediately in a plastic bag and stored at 4’C, they can be
used for 2 wk. Whole blood or blood lysed by saponm (7%) is incorporated m all
media, and must be thoroughly mixed with the liquid agar to ensure an even
distribution. Blood lysed by freezing or thawing is less successful for the growth
of H. pylori.
2. Blood agar bases: Brain-heart mfusion (BHI) agar (Oxoid CM375B; BBL 99069
[Cockeysville, MD]; Difco 0418); Brucella agar (Oxoid CM169; BBL 11088;
Difco 0964); Columbia agar (Oxoid CM33 1; BBL 11125 or Difco 0792).
