Content uploaded by Sarah Umeh
Author content
All content in this area was uploaded by Sarah Umeh on Mar 06, 2019
Content may be subject to copyright.
Nigerian Journal of Microbiology 2016, 30(1): 3299-3303
Published online at www.nsmjournal.org 3299
Faecal Occult Blood Test in Helicobacter Pylori Positive Patients
With Peptic and Gastric Disturbances
Chika Paulinus Enwuru1 and Sarah I. Umeh2
1Imo State College of Nursing and Health Sciences, Amaigbo, Imo State, Nigeria
2Department of Microbiology, Federal University of Technology, Owerri, Nigeria, Phone no 08063955871
Abstract: Helicobacter pylori infection has been variously associated with the risk for a wide spectrum of clinical
outcomes. The infection induces gastric and duodenal ulceration or gastric cancer. Individuals infected with H.
pylori have a 10 to 20% lifetime risk of developing peptic ulcers and a 1 to 2% risk of acquiring stomach cancer.
Over 80% of individuals infected with the bacterium are asymptomatic and it is also believed to play an important
role in natural stomach ecology. In this study, 150patients with gastrointestinal disturbances were subjected to tests
for antibodies to H. pylori and faecal occult blood in order to determine the co-presence of H. pylori infection and
ulcer. Faecal occult blood test was performed using Jonimedics (JDL) occult blood test kit while H. pylori Rapid
Test Device (whole blood/serum/plasma) was used. 13 (8.67%) were positive for Faecal occult blood only, 9 (6%)
were positive for H. pylori only while 55 (36.67%) were positive for both H. pylori infection and faecal occult blood
test. We conclude that faecal occult blood is significantly associated with the presence of H. pylori infection (p=
0.022).
Key words: H. pylori, Occult blood, Ulcer, Gastritis, Pathogenicity.
Introduction
elicobacter pylori, previously
named Campylobacter pylori, is a Gram-
negative, microaerophilic bacterium found
in the stomach. The organism has been
found in other parts of the body, such as the eye
(Giusti,2004, Ahnouxet al, 2004, Cotticelliet al, 2006).
Helicobacter pylori infection hasimportant worldwide
public health significance with an estimated 50% of
the human population being chronically infected by
this pathogen (Luz-del, et al, 2009).Some of the
conditions where H. pylori is present include patients
with chronic gastritis and gastric ulcers which are
conditions that were not previously associated with
a microbial cause. It is also linked to the development
of duodenal ulcers and stomach cancer. Although
Helicobacter pylori infection increases the risk for a
wide spectrum of clinical outcomes, the infection
induces gastric and duodenal ulceration or gastric
cancer in only a minority of infected subjects. Over
80% of individuals infected with the bacterium
are asymptomatic and it is also believed to play an
important role in the natural stomach ecology. Because
up to half the world's population is infected by the
bacterium, it is about the most widespread infection in
the world (Blaser, 2006, Pounder and Ng, 1995,
Bytzeret al, 2011). Actual infection rates vary from
nation to nation; the developing world being more
infected than the industrialized world, where rates are
estimated to be around 25% (Pounder and Ng, 1995).
Acute infection may present as
acute gastritis with abdominal pain (stomach ache)
*Corresponding author:
globachik@yahoo.com Chika Paulinus Enwuru1
Copyright © 2016 Nigerian Society for Microbiology
or nausea (Butcher, 2003). This may develop into
chronic gastritis, with symptoms of non-
ulcer dyspepsia: stomach pains,
nausea, bloating, belching, and sometimes vomiting or
black stool (Ryan, 2010). Individuals infected with H.
pylori have a 10 to 20% lifetime risk of
developing peptic ulcers and a 1 to 2% risk of
acquiring stomach cancer (Chang and Parsonnet, 2010,
Kusterset al 2006).Inflammation of the pyloric
antrum is more likely to lead to duodenal ulcers, while
inflammation of the corpus (body of the stomach) is
more likely to leadto gastric ulcers and gastric
carcinoma (Suerbaum and Michetti, 2002). H.pylori
presence brings about the presence of phagocytic,
inflammatory and immune cells in the gastric lamina
propria (Kim and Moss, 2008), a condition referred to
as chronic gastritis. Pathologists rightly see the
condition as pathological whereas biologists refer to it
as physiological response to indigenous microbiota
(PRIM). Ulcers in the stomach and duodenum result
when the consequences of inflammation allow
stomach acid and the digestive enzyme pepsin to
overwhelm the mechanisms that protect the stomach
and duodenal mucous membrane.
H. pylori virulence factors include
Cag pathogenicity island that contains over 40 genes.
This pathogenicity island is usually absent from H.
pylori strains isolated from humans who are carriers
of H. pylori but remain asymptomatic (Baldwin et al,
2007).The cagA gene codes for one of the
major H.pylori virulence proteins, the cytotoxin-
associated gene A (CagA). Bacterial strains with
the cagA gene are associated with an ability to
cause ulcers (Broutetet al, 2001).H. pylori uses it for
the translocation of the CagA protein to the inside of
the gastric epithelial cells, where it is phosphorylated
in different tyrosine phosphorylation motifs (TPMs).
H
*Enwuru et al.2016 Nigerian Journal of Microbiology, Vol. 30(1): 3299-3303
Published by Nigerian Society for Microbiology 3300
Cytotoxin associated gene CagAcause inflammation
and is potentially a carcinogen (Hatakeyama and
Higashi, 2005).
Vacuolatingcytotoxin A (VacA) is another
virulence factor that is actively secreted into the
adjacent tissue where it produces gastric epithelial
damage (Czajkowsky, et al, 1999).The vacA gene is
present in all H. pylori strains, but the active toxin is
produced by 50% isolated bacteria from clinical cases
and is epidemiologically associated with various
gastroduodenal diseases. It damages epithelial cells,
disrupts tight junctions and causes apoptosis (Smoot,
1997).
The Helicobacter pylori neutrophil-activating
protein (HP-NAP) is a multimeric protein of 150 kDa
that is present in all strains but with a variable level of
expression. HP-NAP exhibits chemiotactic properties
for neutrophils and monocytes and contributes
substantially to their massive infiltration, high
production of reactive oxygen radicals, and adhesion
to gastric endothelium cells, which contributes to the
chronic inflammation of the gastric mucosa (Dundonet
al, 2001, Polenghiet al, 2007).
Urease: H pylori produce the enzyme urease as ureA
and ureB both of which are 6 protein sub-units that
catalyse the hydrolysis of urea to ammonia to
neutralize the acid pH of the mucosa. This enables the
bacteria to evade the Hydrochloric acid of the stomach
and enhance colonization (Meyer-Rosberget al, 1996).
Mutant strains of H. pylori negative for this enzyme
are incapable of infecting the gastric epithelium
(Tsudaet al, 1994).The ammonia may also erode the
mucus barrier as it is toxic to epithelial cells, as are
other biochemicals produced by H. pylori such
as proteases.
In this study, patients with gastrointestinal
disturbances were subjected to tests for antibodies to
H. pylori and faecal occult blood in order to determine
the co-presence of H. pylori infection and ulcer. Faecal
occult blood test is routinely performed to detect
bleeding in the upper gastrointestinal tract as
indication of gastric and duodenal ulcers. Blood from
these lesions is usually broken down or digested
before passing out in faeces as occult blood.
Conversely, blood from lower gastrointestinal
bleeding pass out as intact red blood cells and can be
seen with the naked eyes or microscopically.
Methodology
Sample Collection
Preparation of Patients
Prior to the faecal occult blood test, the
patients were instructed to avoid the intake of
the following, 3-7 days before the test, which,
according to Fletcher (2001), could give false
positive results:
Certain fruits, especially prunes, grapes,
plums, and apples.
Generous amounts of both cooked and uncooked
vegetables, including lettuce, corn, spinach, and
celery.
Red meat
Vitamin C and iron supplements
Non-steroidal anti-inflammatory drugs
Rectal Preparations.
Patients with known lower GIT bleeding and
hemorrhoids and women on menstrual flow were
excluded from the study.Stool samples were collected
in clean, dry specimen containers with wooden
applicator.
Blood samples were collected in dry anti-coagulant
free specimen container and allowed to clot. The
clotted blood was centrifuged at 3000 revolutions per
minute and the serum separated using Pasteur pipette.
Procedure for Testing
Faecal Occult Blood
Faecal occult blood test was performed using
JDL occult blood test kit (Jonimedics, Enugu,
Nigeria). Haemoglobin or its iron derivatives catalyze
the oxidation of a non-carcinogenic chromogen (JDL
chromogen) to a pink colour in the presence of
peroxide.
The kit comprises of
Solution A
Solution B
Thick glossy paper
Buffer
A little portion of the stool sample was
emulsified in 2 drops of the buffer on the glossy
paper.Positive control was prepared by adding 0.05 ml
of blood to 2 litres of distilled water. The buffer was
used as negative control. Two drops of each control
was put on separate glossy paper and treated the same
way as the test samples.A drop of solution A was
added and followed by a drop of solution B.It was
examined for development of colour at 2 minutes.
Interpretation of Result
Development of pink colour indicates positive
test for occult blood while absence of pink colour
indicates negative test for faecal occult blood (Rockey,
1999).
Note: The reaction with intact red cells probably from
lower GIT bleeding occurs very slowly while the
reaction with digested blood (as in ulcer conditions) is
rapid. Therefore the result should not be read beyond 2
minutes.
H. pyloriRapid Diagnostic Test
H. pylori Rapid Test Device (whole
blood/serum/plasma) was used for the test. It is a
qualitative membrane device based immunoassay for
the detection of H. pylori antibodies in whole blood,
*Enwuru et al.2016 Nigerian Journal of Microbiology, Vol. 30(1): 3299-3303
Published by Nigerian Society for Microbiology 3301
serum or plasma using a combination of H. pylori
antigen coated particles and anti-human IgG.
Specimen followed by buffer is added to the specimen
well of the test device. The specimen migrates
chromatographically along the length of the test strip
contained within the device and interacts with the
reagents on the strip. If the specimen contains H.
pylori antibodies, a coloured line will appear in the test
line region indicating a positive result. If the specimen
does not contain H. pylori antibodies, a coloured line
will not appear in the test region (Pronovostet al,
1994).
The kit contains the following:
Test devices (in sealed pouches)
Disposable specimen droppers
Buffer (for whole blood only)
The test devices, the buffer, the controls and the
specimen were allowed to equilibrate at room
temperature for at least 30 minutes before testing.The
test device was removed from the pouch and placed on
a level surface.With the disposable specimen dropper,
4 drops of the serum (about 100µl) was delivered into
the specimen well (S) of the test device. A timer was
started.The device was examined for the appearance of
red lines after 10 minutes.
Interpretation of Results
If two distinct red lines appear, one on the test
region and the other on the control region, the test is
positive for antibodies to H. pylori.If one red line
appears only on the control region and not on the test
region, the test is negative.If the control line fails to
appear, the test is invalid irrespective of whether a red
line appears on the test region or not.
Results
A total of 150 patients, 79 males and 71
females, were tested for H. pylori infection and faecal
occult blood from 2011 to 2013 (see table 1). Out of
this number, 13 (8.67%) were positive for Faecal
occult blood only, 9 (6%) were positive for H. pylori
only while 55 (36.67%) were positive for both H.
pylori infection and faecal occult blood test.
Table 1: Sex Distribution with H. pylori infection associated with positive/negative faecal occult blood
Year No tested H.pylori-/FOB+ H.pylori+/FOB- H.pylori+
FOB + M F M F M F M F
2011 20 28 2 2 2 1 8 8
2012 25 28 2 3 1 1 9 11
2013 34 15 2 2 3 1 11 8
Total 79 71 6 7 6 3 28 27
On the age group distribution, Figure 1 shows that 31-40 years and 51 -60 years have the highest prevalence
rate of 15 out of 30 patients and 15 out of 25 patients tested in those age groups respectively.
The older age groups were more positive for faecal occult blood in the absence of H. pylori infection whereas
the younger age groups were more positive for H. pylori infection with negative faecal occult blood test.
Figure 1: Age Distribution with H. pylori infection associated with positive/negative faecal occult blood
Discussion, Conclusion and Recommendation
In this study, 150 patients were subjected to
tests in order to find the correlation between infection
with H. pylori and the development of upper
gastrointestinal ulcers. Seventy seven (51.3%) of those
investigated were either positive for H. pylori infection
*Enwuru et al.2016 Nigerian Journal of Microbiology, Vol. 30(1): 3299-3303
Published by Nigerian Society for Microbiology 3302
without the presence of faecal occult blood, positive for
faecal occult blood in the absence of antibodies to H.
pylori or positive for faecal occult blood in the presence
of H. pylori infection. Precisely, 36.67% of the study
samples were positive for both H. pylori and faecal
occult blood tests while 42.67% were positive for H.
pylori. This is close to the report that almost 50% of the
world population was infected with H. pylori (Pounder
and Ng, 1995). There was a strong correlation between
H. pylori infection and positive faecal occult blood
hence upper gastrointestinal ulcer. Fifty five patients
(71.4%) out of the 77 that have positive results were
positive for both H. pylori infection and faecal occult
blood.Earlier researches have associated the presence of
H. pylori in the gastric lumen with the presence inthe
gastric lamina propria of phagocytic and immune cells,
a condition known as ‘chronic gastritis’as reported by
Warren, (1983), Marshall and Warren, (1984) with
subsequent increased risk for development ofpeptic
ulceration (Nomura, et al, 1994, Nomura et al,2002)
and gastric adenocarcinomaand lymphoma. Ulcers in
the stomach and duodenum result when the
consequences of inflammation allow stomach acid and
the digestive enzyme, pepsin to overwhelm the
mechanisms that protect the stomach and
duodenal mucous membranes.
There is no significant difference between
male and female H. pylori infection and positive faecal
occult blood test (p= 0.049). The middle age groups
from 31-60 years were most infected with H. pylori and
have most positive faecal occult blood. Infection within
this middle age (31 years) and tending towardsold age
(60s) could stillreflect higher infection rates in the past
when the individuals were children rather than more
recent infection at a later age of the individual as
previously reported (Kusterset al, 2006). It was further
posited that infections are usually acquired in early
childhood in all countries (Kusterset al, 2006). In our
study those within the younger ages from 10 years and
up to 50 years have more of H. pylori infection with
negative faecal occult blood test compared to the older
age groups. However, Pounder et al, (1995) reports that
the percentage of infected people increases with age,
with about 50% infection for those over the age of 60
compared with around 10% between 18 and 30
years.This probably could be as a result of declining
immunity due to aging. Brown (2000) stated that the
age at which this bacterium is acquired seems to
influence the possible pathologic outcome of the
infection: people infected with it at an early age are
likely to develop more intense inflammation that may
be followed by atrophic gastritis with a higher
subsequent risk of gastric ulcer, gastric cancer, or both.
Acquisition at an older age brings different gastric
changes more likely to lead to duodenal ulcer (Pounder
and Ng, 1995).Individuals infected with H. pylori have
a 10 to 20% lifetime risk of developing peptic
ulcers and a 1 to 2% risk of acquiring stomach cancer
(Chang and Parsonnet, 2010, Kusterset al, 2006).
Furthermore, the older age group in our study
have more faecal occult blood positive in the absence of
H. pylori infection. This could be as a result of ulcer
from other causes other than H. pylori infection or that
H. pylori infection could have been eradicated by
antibiotic therapy, leaving behind the consequence of
its earlier presence which could take some time to
resolve. We conclude that 71.4% of the patients that
were either positive for H. pylori infection or faecal
occult blood test were positive for both tests and this
shows a positive correlation between the presence of H.
pylori infection and ulcer in the upper gastrointestinal
tract.
We recommend that for patients suspected of
ulcer in the upper GIT, tests to identify the presence of
H. pylori infection be conducted and appropriate
antimicrobial therapy instituted as part of chemotherapy
for peptic and duodenal ulcers.
References
Ahnoux-Zabsonre A, Quaranta M, Mauget-Faÿsse M (2004).
"Prévalence de l'Helicobacterpyloridans la
choriorétinopathieséreusecentraleetl'épithéliopathier
étinienne diffuse" [Prevalence of Helicobacter
pylori in central serous chorioretinopathy and
diffuse retinal epitheliopathy: a complementary
study]. Journal Françaisd'Ophtalmologie (in
French) 27 (10): 1129–33.
Baldwin DN, Shepherd B, Kraemer P, Hall MK, Sycuro LK,
Pinto-Santini DM, Salama NR (2007). Identification
of Helicobacter pylori Genes That Contribute to
Stomach colonization.Infect. Immun. 75 (2): 1005–
16.
Blaser MJ (2006). ‘Who are we? Indigenous microbes and the
ecology of human diseases’ EMBO Reports. 7 (10):
956–60.
Broutet N, Marais A, Lamouliatte H, de Mascarel A,
Samoyeau R, Salamon R, Mégraud F. (2001) CagA
Status and Eradication Treatment Outcome of Anti-
Helicobacter pylori Triple Therapies in Patients
with Nonulcer Dyspepsia. J.Clin. Microbiol. 39 (4):
1319– 22.
Brown LM (2000)."Helicobacter pylori: epidemiologyand
routes of transmission". Epidemiol. Rev. 22 (2):
283–97.
Butcher, GP. (2003). Gastroenterology: An Illustrated Colour
Text. Elsevier Health Sciences.p. 25. ISBN 0-443-
06215-3.
Bytzer P, Dahlerup JF, Eriksen JR, Jarbøl DE, Rosenstock S,
Wildt S. (2011)."Diagnosis and treatment of
Helicobacter pylori infection". Dan.
Med.Bul.l 58 (4): C4271. Retrieved 7 August, 2013.
Chang, AH.,Parsonnet, J. (2010)."Role of Bacteria in
Oncogenesis". Clinical Microbiology
Reviews 23 (4): 837–857.
Cotticelli L, Borrelli M, D'Alessio AC, Menzione M, Villani
A, Piccolo G, Montella F, Iovene MR, Romano M
(2006). "Central serous chorioretinopathy and
Helicobacter pylori" European journal of
ophthalmology 16 (2): 274–8.
Czajkowsky DM, Iwamoto H, Cover TL, Shao Z. (1999) The
vacuolating toxin from Helicobacter pylori forms
*Enwuru et al.2016 Nigerian Journal of Microbiology, Vol. 30(1): 3299-3303
Published by Nigerian Society for Microbiology 3303
hexameric pores in lipid bilayers at low pH.Proc.
Natl. Acad. Sci. USA; 96 (5): 2001-2006.
Dundon WG; Nishioka H; Polenghi A; Papinutto E; Zanotte
G, MontemurroP,DelGiudice G, RinoRappuoli R
and Montecucco C. (2001). The neutrophil-
activating protein of Helicobacter pylori. Int. J.
Med.Microbiol.; 291: 545-550.
Fletcher RH. (2001) Diet for Fecal Occult Blood Test
Screening: Help or Harm? Effective Clinical
Practice;4:180-182.
Giusti C. (2004). "Association of Helicobacter pylori with
central serous chorioretinopathy: Hypotheses
regarding pathogenesis". Medical
Hypotheses 63 (3): 524– 7
Hatakeyama M, Higashi H. (2005). Helicobacter pyloriCagA:
A new paradigm for bacterial
carcinogenesis". Cancer Science, 96 (12): 835–843.
Kim, W. & Moss, SF. (2008). "The role of H. pylori in the
development of stomach cancer". Oncology
Review. 1 (Suppl 1): 165–168.
Kusters JG, van Vliet AH, Kuipers EJ.(2006). ‘Pathogenesis
of Helicobacter pylori infection’.Clin.Microbiol.
Rev. 19 (3): 449–490.
Luz del C, Hernández-H, Eduardo C, Lazcano P, Yolanda
LV, Germán R, Aguilar-G. (2009) Relevance
of Helicobacter pylori virulence factors for vaccine
development Salud. pública.
Méx. vol.51, suppl.3 Cuernavaca.
Marshall BJ, Warren JR. (1984) Unidentified curved bacilli
in the stomach of patients with gastritis and peptic
ulceration. Lancet; 1:1311–1315.
Meyer-Rosberg K, Scott DR, Rex D, Melchers K, Sachs G.
(1996) The effect of environmental pH on the
proton motive force of Helicobacter
pylori. Gastroenterology; 111:886-900.
Nomura A, Stemmermann GN, Chyou PH, Perez-Perez GI,
Blaser MJ.(1994).Helicobacter pylori infection and
the risk for duodenal and gastric
ulceration.Ann.Intern.Med; 120:977–981.
Nomura A.M.Y, Perez-Perez G.I, Lee J, Stermmermann G
and Blaser M. J. (2002).Relationship between H.
pyloricagA status and risk of peptic ulcer
disease.Am.J.Epidemiol; 155:1054–1059.
Polenghi A, Bossi F, Fischetti F, Durigutto P, Cabrelle A,
Tamassia N, Cassatella MA, Montecucco C,
Tedesco F, de Bernard M.(2007)The neutrophil-
activating protein ofHelicobacter pylori crosses
endothelia to promote neutrophil adhesion in vivo.
The J. of Immun; 178:1312–1320.
Pounder RE, Ng D. (1995). "The prevalence of Helicobacter
pylori infection in different countries" Aliment.
Pharmacol.Ther. 9 (Suppl 2): 33–39.
Pronovost AP., Rose SL., Pawlak J, Robin H and Schneider
R. (1994) Evaluation of a new immunodiagnostic
assay for Helicobacter pylori antibody detection:
Correlation with histopathological and
microbiological results. Journal of Clinical
Microbiology, 32: 46-50
Ryan, K. (2010). Sherris Medical Microbiology.McGraw-
Hill.pp. 573, 576.ISBN 978-0-07-160402-4.
Rockey DC. (1999) Occult gastrointestinal bleeding. New
England Journal of Medicine, 341 (1): 38-46
Smoot DT (1997). "How does Helicobacter pylori cause
mucosal damage? Direct
mechanisms". Gastroenterology 113 (6 Suppl):
S31–34; discussion S50.
Suerbaum S, Michetti P. (2002). "Helicobacter
pylori infection". N. Engl. J. Med. 347 (15): 1175–
1186.
Tsuda M, Karita M, Morshed MG, Okita K, Nakazawa T.
(1994) A urease-negative mutant of Helicobacter
pylori constructed by allelic exchange mutagenesis
lacks the ability to colonize the nude mouse
stomach. Infect Immun; 62:3586-3589.
Warren JR. (1983) Unidentified curved bacilli on gastric
epithelium in active chronic gastritis. Lancet;
1:1273.
