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2 | Primary isolation
and detection of Helicobacter pylori from dyspeptic patients: a simple, rapid method

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authors pylori from dyspeptic patients: a simple, rapid
Information for
method
reviewers A. Al-Sulami,1 H.S. Al-Kiat,2 L.K. PDF version
Bakker1 and H. Hunoon3
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Helicobacter ‫ا=ستفراد البدئي وكشف جرثومة الـمَ لوي(ة البوابية‬
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‫ طريقة سريعة وبسيطة‬:‫عدية‬C‫ في مرضى القرحة ا‬pylori
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‫ حنون‬%‫ حس‬،‫ياء كاظم باقر‬1 ،‫ هاشم الخياط‬،‫ عبد الجبار السلمي‬%‫أم‬
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‫ استهدفت هذه الدراسة استنباط طريقة سريعة وبسيطة‬:‫صـة‬I‫الخ‬
Disclaimer/copyright Helicobacter pylori ‫ة البوابية‬P‫الـملوي‬
َ ‫ستفراد البدئي وكشف جرثومة‬Z‫ل‬
‫خاطي‬1‫ من الغشاء ا‬،‫نظار‬1‫ با‬،‫ وقد أخذت خزعتان‬.‫عدية‬1‫في مرضى القرحة ا‬
Contact us ‫ مريضا ً شخصت إصابتهم على أنها قرحة‬136 ‫للمنطقة البوابية للمعدة من‬
‫ل الفحص‬Z‫ ومن خ‬.‫ في العراق‬،‫ وذلك في مستشفى البصرة العام‬،‫هضمية‬
ً ‫ مريضا‬81 ‫ة البوابية في‬P‫الـملوي‬
َ ‫ تم التعرف على‬،‫الهيستوباثولوجي للخزعات‬
‫ فقد استُنبتت‬،‫ أما بالنسبة للزرع الجرثومي‬.(%59.6) ‫عدية‬1‫بالقرحة ا‬
‫ انتقائي استخدم فيه وسط‬- ‫ول غير‬x‫ ا‬:%‫ متوازي‬%َ‫ستَنْبت‬ ْ ‫الخزعات في ُم‬
‫ وكان ا{خر انتقائيا ً استخدم فيه أغاريوريا كولومبيا‬،‫كولومبيا التقليدي‬
‫عدل الناتج من استخدام‬1‫ستفراد أعلى من ا‬Z‫ الذي أظهر معد~ً ل‬.‫ور‬P ‫ح‬1‫ا‬
‫ كما أن‬،(‫رضى‬1‫ من ا‬%44.1 ‫ مقابل‬%67.6) ‫أغار كولومبيا التقليدي‬
‫ ساعة مقابل خمسة إلى سبعة أيام( مع‬24) ‫النتائج ظهرت بصورة أسرع‬
.‫ي شك‬x ً~‫صورة أوضح ~ تترك مجا‬

ABSTRACT: The study aimed to develop a rapid and

simple method for the primary isolation and detection of
Helicobacter pylori from dyspeptic patients. Mucosal
:
 antral biopsy specimens were obtained from 136
consecutive dyspeptic patients diagnosed with peptic
ulcer by endoscopy at Basra General Hospital, Iraq. From
histopathological examination of biopsies, H. pylori was
detected in 81 (59.6%) peptic ulcer patients. For bacterial
culture, specimens were cultured in parallel on 2 media:
the non-selective classic Columbia agar and the selective
modified Columbia urea agar (MCUA). MCUA showed a
higher isolation rate than classic Columbia agar (67.6%
versus 44.1% of patients), and the results were obtained
faster (24 hours versus 5–7 days) with more clear-cut
identification.

Méthode rapide et simple d’isolement primaire et de

détection de Helicobacter pylori chez des patients
dyspeptiques

RÉSUMÉ: L’objectif de cette étude était de mettre au

point une méthode rapide et simple permettant d’isoler et
de détecter Helicobacter pylori chez des patients
dyspeptiques. Des échantillons de muqueuse ont été
prélevés grâce à une biopsie antrale sur 136 patients
dyspeptiques consécutifs pour lesquels un diagnostic
d’ulcère gastro-duodénal avait été établi par endoscopie
à l’hôpital général de Bassora (Basra) en Iraq. L’examen
histopathologique des biopsies a permis de détecter H.
pylori chez 81 (59,6 %) patients atteints d’un ulcère
gastro-duodénal. Concernant la culture bactérienne, les
échantillons ont été mis en culture en parallèle dans deux
milieux : la gélose Columbia classique non sélective et la
gélose Columbia sélective modifiée à l’urée. Cette
dernière a présenté un taux d’isolement plus élevé que la
gélose Columbia classique (67,6 % des patients contre
44,1 %) et les résultats ont été obtenus plus rapidement
(24 heures contre 5 à 7 jours), avec une identification
plus nette.

1 Department of Biology, College of Education, University

of Basra, Basra, Iraq (Correspondence to A. Al-Sulami:
[email protected]).
2 Department of Surgery;3 Department of Laboratories,

Basra General Hospital, Basra, Iraq

Received: 03/10/05; accepted: 12/02/06
EMHJ, 2008, 14(2): 268-276
:
 Introduction
Peptic ulcer disease is a common problem encountered
by physicians in everyday practice. Since the isolation of
Helicobacter pylori by Marshall and Warren in 1983 [1],
tremendous progress in the understanding of the etiology,
pathogenesis and management of this disease has
occurred.

It is now widely agreed that H. pylori is the cause of most

peptic ulcer disease [2,3] and that this microorganism is
endemic in some developing countries, affecting as much
as half of the population [4]. The strongest evidence for
the pathogenic role of H. pylori in peptic ulcer disease
comes from treatment trials. Eradication of the organism
results in ulcer healing and reduces the risk of ulcer
recurrence and complications [5,6].

Infection by H. pylori has been diagnosed by a variety of

invasive and non-invasive tests [7]. However, the “gold
standard” for H. pylori detection, as suggested by the
Maastricht consensus report [8] is positive culture or both
a positive histologic examination and a positive rapid
urease test. The sensitivity of histology is generally high;
however, because H. pylori colonization is focal, negative
biopsy results do not exclude the possibility of infection in
areas not sampled [9].

A variety of culture media have been used for isolation of

H. pylori. Both selective and non-selective media have
been used [10,11] . The most commonly used are
Columbia, Brucella, brain–heart infusion, trypticase soy or
blood agar media, each supplemented with 5%–10%
blood. Supplementation of culture media with serum,
albumin or activated charcoal instead of blood has been
used to support the growth of H. pylori [12]. These
materials are assumed to play a role of detoxifying the
toxic substances in the media [13]. Bacterial growth
usually appears as translucent, small, pinpoint colonies
[1,14].

We aimed to develop a rapid and simple method for the

primary isolation and detection of H. pylori from dyspeptic
patients.

Methods
:
 Sample

Between October 2002 and August 2004, 136 patients

with symptoms suggestive of peptic ulcer were diagnosed
as having peptic ulcer (gastric and duodenal) using
endoscopic examination at the endoscopic unit, Basra
General Hospital, Iraq, by a specialized surgeon. There
were 72 males and 64 females. The ages of the patients
ranged from 18 years to 69 years. Cases with negative
endoscopic results for peptic ulcer were excluded from
the study.

Data collection

Two gastric tissue specimens were taken from the antral

region of the stomach of each patient during the
endoscopic examination. Presence of the bacterium H.
pylori in the tissue specimens was detected using 2
methods: histopathological examination and bacterial
culture growth.

Laboratory methods

For histopathological detection, surgical specimens were

fixed in paraffin wax and stained with haematoxylin and
eosin (H&E) and Giemsa stains. The sections were
examined for the presence of H. pylori and any
inflammatory changes that might be present in the gastric
antral sections using the ordinary light microscope. The
Sydney system was used to assess the histological
changes. In this system the topography of the gastritis as
seen on endoscopy is included (antral, body,
pangastritis), together with the macroscopic appearances
(oedema, haemorrhagic, flat, raised, etc.). In addition,
some of the histological and microscopic features may be
graded in terms of severity as mild, moderate or severe.

For bacterial culture detection, antral biopsy specimens

were transported to the microbiology department within 1
hour in 5 mL tryptic soy broth as a transport medium. In
the laboratory, specimens were first ground in a sterile
mortar with the aid of a sterile fine glass rod until the
formation of a homogenate. Two kinds of culture media—
the classic Columbia agar and the modified Columbia
urea agar (MCUA)—were used. MCUA contains per litre:
41 g Columbia agar base, 10 mL of haemin solution, 20 g
of urea, 0.0012 mg of phenol red and 0.04 mg of
:
 vancomycin. Slants of this medium were prepared in 14
×16 mm test tubes. Each slant tube was made up to
contain 5 mL of MCUA.

Haemin solution was prepared by dissolving 50 mg of

haemin in 1 mL of 1.0 mol NaOH. Distilled water was then
added to make 100 mL solution which was then sterilized
by autoclave at 121 ºC for 15 minutes. The solution was
stored at 4 ºC and used as stock solution.

From the homogenate, 1 mL was taken and placed at the

bottom of the MCUA slant tube so that the transport
medium itself is the liquid phase. The tube was tilted a
few times to allow the added broth homogenate to
moisten the upper slanted portion of the tube before its
settlement into the bottom of the slant. The resulting
system is a simple monophasic–diphasic culture setup
(MDCS); a diphasic solid liquid environment at the bottom
of the test tube and a monophasic solid one above it [15].
At the same time some part of the homogenized biopsy
specimen from each patient was spread and plated on
standard Columbia agar medium.

With MCUA medium, the inoculated tubes were incubated

microaerophilically at 37 ºC for 24 hours, after which the
colour changes from orange to pink in the solid phase,
indicating urease activity, and the appearance of isolated
H. pylori colonies was observed. With classic Columbia
agar medium, the incubation time was extended from 5 to
7 days. After that time H. pylori growth was observed as a
few, tiny transparent colonies. The isolated H. pylori
colonies were then subcultured on plates of the same
MCUA medium for purification, identification, and
performing antibiotic susceptibility tests.

Identification of isolated H. pylori was confirmed by a

negative reaction to Gram staining and by positive results
of each of the following biochemical tests: oxidase test,
catalase test and urease test, as indicated by colour
change of the medium from orange to pink [16].

Results
Of the total 136 patients with positive endoscopic
diagnosis of peptic ulcer, 92 patients (67.6%) showed
positive evidence of H. pylori infection using bacterial
culture and 81 patients (59.6%) histopathologically. They
:
 were referred to as H. pylori ulcer patients. In the
remaining 44 patients (32.4%), called non-H. pylori ulcer
patients, H. pylori were not detected.

Patient characteristics

Among H. pylori ulcer patients, the highest detection rates

of the bacterium (79.2%) was recorded in the age group
41–50 years, while no single case was recorded in the
age group ≤ 20 years (Table 1). These findings were
statistically nonsignificant (χ2 = 0.733, P > 0.05, df = 4).

Of the 72 males with peptic ulcer, 49 (68.1%) showed

positive tests for H. pylori, while 43 (67.2%) out of 64
females in this study showed positive tests for H. pylori
(Table 1).

The highest occurrence of H. pylori was recorded in

patients of low educational status. The bacterium was
detected in 74.1% (40 out of 54) of these peptic ulcer
patients with low educational level compared with 58.3%
(21 out of 36) with high educational level but this was not
statistically significant (χ2 =2.447, P > 0.05, df = 2).

A higher rate of H. pylori was recorded in patients from

rural areas. In 68.5% (50 out of 73) of patients living in
rural areas H. pylori was detected compared with 66.7%
(42 of 63 ulcer patients) in urban areas; again these
results were statistically not significant (χ2 = 0.04, P >
0.05, df = 1).

The habit of cigarette smoking was reported by 55.1% of

all ulcer cases. Among cigarette smokers 55 out of 75
(73.3%) had H. pylori infection compared with only 60.7%
(37 out of 61) of nonsmoker peptic ulcer patients, a
statistically non-significant finding.

Histopathology findings

For histopathological detection of H. pylori, the

distribution of H. pylori was examined in both H&E-
stained and Giemsa-stained sections. The changes in
background mucosa included inflammation, atrophy and
intestinal metaplasia. The degree of inflammatory
changes in the gastric biopsy specimens were evaluated
in every H. pylori ulcer patient. Table 2 shows that the
most frequent pattern of inflammatory changes of antral
:
 biopsy specimens from patients with H. pylori ulcers was
severe gastritis, recorded in 41 patients (44.6%), followed
by mild gastritis in 31 patients (33.7%) and chronic
gastritis in 20 patients (21.7%).

Culture findings

The total isolation rate of H. pylori was about 44.1% by

the classic Columbia agar, as it identified 60 positive
infections out of 136 patients tested. The growth of H.
pylori on this medium was scanty, the colonies were few
in number, transparent and tiny in size. Bacterial
contamination of the medium was frequent. The
contaminant bacteria were Pseudomonas spp., Proteus
spp. and Klebsiella spp. The growth rate of H. pylori on
classic Columbia agar was slow; in most cases 5 to 7
days were needed for the colonies to appear.

The MCUA using MDCS showed a greater isolation rate

of H. pylori. Positive results were found in 92 out of 136
ulcer patients (67.6%). The colonies of the isolated
bacteria were abundant in number, creamy in colour and
larger in size, about the size of a pinhead, when
compared with the colonies on the classic Columbia agar.
There was no contamination at all. The isolation was very
rapid; only 24 hours were needed for the growth to be
identified. The change in colour of the slanted medium
from orange to pink caused by urease action occurred at
the same time, giving additional evidence for the
presence of H .pylori in the tissue specimens.

All H. pylori isolates were subjected to Gram staining. The

characteristics of this bacterium were observed as Gram-
negative, spiral shaped rods.

The 3 additional biochemical tests (oxidase, catalase and

urease) performed to confirm the identity of H. pylori were
positive for all 92 isolates.

Discussion
The discovery of H. pylori by Warren and Marshall in
1983 has changed the conventional concept of
gastroduodenal ulcer disease [1]. Studies over the years
suggest a high correlation between H. pylori infection and
peptic ulceration [17]. The prevalence of H. pylori
infection shows a wide geographical variation [18,19]. It is
:
 reported that 60% to 70% of patients with gastric ulcer,
and 90% to 95% of patients with duodenal ulcer, have
marked gastric colonization of H. pylori [20].

Although it cannot be said that H. pylori causes

ulceration, as half of the healthy population also harbours
this organism, it has been shown that infection certainly
makes the occurrence of ulcers more likely [20,21]. Also,
eliminating this bacterium reduces the rate of ulcer
recurrence to less than 25% [5].

In Iraq, several studies have been conducted to evaluate

the prevalence of H. pylori infection in peptic ulcer
disease indicating a range of 60%–70 % [22–24].

The major anatomical focus of this Gram-negative

microaerophilic bacterium is the gastric antrum, the
narrower lower part of the stomach, and also the
duodenum when the normal type of duodenal epithelium
is replaced with antral type mucosa. Helicobacter will
infect this antral type of mucosa [20,25]. The results of
our study also support this concept, since all positive
cases for H. pylori were identified in antral biopsy
specimens.

Inflammatory changes in antral biopsy specimens were

found in all H. pylori ulcer patients in this study. These
changes were mild gastritis in 33.7%, severe gastritis in
44.6%, and chronic gastritis in 21.7% of H. pylori ulcer
patients. Identification of H. pylori as Gram-negative
spiral-shaped organisms in stained antral biopsy
specimens was possible in only 81 out of 92 H. pylori
ulcer patients. The possible explanation for the remaining
11 cases (false-negative results) may be due to the
patchy distribution of the organism in the antral part of
stomach in peptic ulcer disease [9].

The gold standard for the presence of most infectious

disease is successful culture of the microorganism [8]. At
present, culture of H. pylori from gastric antral biopsy
specimens is a reference technique in bacteriology and is
essential for drug susceptibility testing and analysis of
putative virulence factors [12,26]. Although it is usually
considered a tedious, time-consuming and expensive
procedure, culturing on solid medium is the standard
technique used in most laboratories for the isolation of H.
:
 pylori from gastric biopsy specimens [11].

Primary isolation of H. pylori from a biopsy specimen is a

difficult process, the typical success rates in culturing the
organism are reported to be in the range of 70% to 80%
with 90% to 95% sensitivity and 100% specificity [20].
Several factors, which are difficult to control, cause
difficulty with the culturing of the organism: patchy
distribution of the organism on the gastric mucosa,
contamination of biopsy forceps, presence of
oropharangyeal flora, loss of viability of the organism
during transportation, etc. All these may be responsible
for a poor negative predictive value associated with
culture of H. pylori [4,27].

In the present study the isolation rate of H. pylori by

bacterial culture method in 136 peptic ulcer patients was
67.6%. This rate is comparable to the above mentioned
international results.

A variety of media, selective and non-selective, or a

combination of both, have been proposed for use in the
primary isolation of H. pylori, but the optimal method of
recovery still remains to be established [28]. Columbia
blood agar is a non-selective medium used for many
years alone or in combination with other non-selective
and selective media for culturing H. pylori from antral
biopsy specimens taken from peptic ulcer patients during
upper gastrointestinal endoscopy [11,29]. The isolation
rate of H. pylori using this medium alone is very variable.
Results as low as only 28.5% total isolation rate were
reported by some authors [30]. The isolation rate of H.
pylori using Columbia blood agar in the present study was
44.1%; the colonies were few in number and tiny in size.
Bacterial contamination of the medium was frequent. The
contaminant bacteria were Pseudomonas spp., Proteus
spp. and Klebsiella spp ., the source of which could be
contaminated biopsy forceps and contamination during
obtaining, transporting and preparing of the defibrinated
sheep blood added to the classic Columbia agar. The
growth rate of H. pylori on this medium was slow, as 5 to
7 days were needed for the colonies to appear.

Efforts have been invested to improve the reliability of

Columbia agar and change it to become selective for H.
pylori. Westblom et al. in 1991 described egg yolk
:
 emulsion agar (EYE) [10]. When this medium was
compared with other media, the colony count for EYE
agar was significantly higher. However, the results of
using EYE agar alone as a selective medium for culturing
H. pylori in gastric biopsy specimens in peptic ulcer are
controversial. While the maximum isolation rate was less
than 51% in one study, another study stated that adding
egg yolk emulsion to the culture medium was significantly
worse than the use of whole blood [31].

Our efforts to establish an optimal method for the

recovery of H. pylori from antral biopsy specimens in
peptic ulcer patients is based on a modification of the
Columbia agar medium to make it selective for culturing
H. pylori. Many reports also mentioned that the use of
selective and non-selective media in parallel is superior to
the use of one medium alone [10,11,30,31]. By comparing
these results of H. pylori isolation rates with our results, it
is clearly evident that the 67.6% H. pylori isolation rate
obtained in the present study is reasonably high. Using
MCUA is much superior to using a combination of
different selective and non-selective media in parallel to
culture H. pylori. The high H. pylori isolation rate with
MCUA may be attributed to the use of haemin as a sole
iron source, which proved to be superior to other iron
sources such as whole blood or serum. Also the clean
procedure of preparing and adding haemin solution to the
modified medium resulted in a total absence of bacterial
contamination, thus removing the antagonistic effect of
co-plated contaminants.

Conclusions
The results obtained with this slanted MCUA medium
were encouraging as they showed great advantages over
many selective media used alone or in combination for
isolating H. pylori. The advantages of such modifications
over Columbia agar medium may be summarized as
follows:

1. High isolation rate of H. pylori, 67.6 %.

2. Rapid culturing of H. pylori as growth of H. pylori is
very clear within 24 hours of incubation instead of 5–
7 days needed with classic Columbia agar. This
could be ascribed to the better accessibility of iron
from haemin as compared to whole blood or
:
 erythrocytes. Also, the lack of contaminant microbes
may enhance faster growth by eliminating the effect
of competition.
3. Colony count is significantly higher, and colonies are
very clear and relatively large in size.
4. Microbial contamination of the medium is totally
absent.
5. Incorporation of urease test in the medium provides
clear evidence for the presence of H. pylori in the
tissue specimen. It is indicated by colour change in
the medium, from orange to pink, due to the splitting
effect of urease enzyme on the urea component of
the modified medium.
6. Using MDCS provides several advantages over
other culture techniques, exemplified by the fast
appearance of results, elimination of transport
medium and establishment of simultaneous but
separate environments for both the isolation and
identification of H. pylori. It also provides flexibility in
the kinds of media used in the liquid and solid
phases. Consequently, the method is inexpensive,
and less time-consuming.

We conclude that preparing this single selective medium

(MCUA) is clearly simpler, less time-consuming, and
much more economic than preparing and working with
multiple culture media in parallel.

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