International Journal of Application or Innovation in Engineering & Management (IJAIEM)
Web Site: www.ijaiem.org Email: editor@ijaiem.org
Volume 4, Issue 7, July 2015
ISSN 2319 - 4847
Detection of Helicobacter pylori in Patients with
Ulcer Using Stool and Serum Antigen Tests
1
Agu Georgia Chinemenwa, 2Salami Olufunmilayo Olamide, 3Agboola Oluwafemi Peter,
4
Ajuazim Kingsley Chinedu
1
Olabisi Onabanjo University, Department of Microbiology, PMB 2002, Ago -Iwoye, Ogun State
2
Science Laboratory Technology, (Microbiology unit) Federal College of Animal Health & Production Technology, PMB
5029, Moor plantation Ibadan, Oyo State
3
Olabisi Onabanjo University, Microbiology Department, Ago-Iwoye, Ogun State.
4
Olabisi Onabanjo University, Microbiology Department, Ago- Iwoye, Ogun State
ABSTRACT
Helicobacter pylori are one of the most common infections in humans, with an estimated 50% of the world’s population being
infected (Torres et al, 2000). The presence of Helicobacter pylori in patients with ulcer disease using stool and serum antigen
tests, antibiotics sensitive profile were investigated, serological methods using antigen kits(Acon San Diego, CA,USA) was
employed in detecting H. pylori. The prevalence of this organism was evaluated among the sex or gender in all the selected
three states. However stool samples that were negative for Helicobacter pylori were further cultured onto MacConkey and
Salmonella-Shigella media, the sensitivity of the isolates to various antibiotics were carried out using agar diffusion method.
One hundred and forty samples each of stool and blood were collected from three locations in south Western Nigeria, It was
observed that 42(30%),98(70%),32(22.8%), and 108(77.1%) patients tested positive and negative for Helicobacter pylori disease
using stool and serum samples respectively. However, samples from Ogun state had the highest prevalence rate of 50%,this was
followed by 29% and 21% rate from Ibadan and Lagos respectively, Cultures of the stool samples revealed the presence of
Klebsiella spp, E coli, Salmonella spp and Shigella spp, with E coli having the prevalence 22(43.1%). The sensitivity of the
isolated organisms varies according to the drug and location with 18mm and 16mm being the highest inhibitory zones for
Lagos, Ibadan and Ogun State respectively. It could therefore be concluded that the serological methods used was reliable to
some extent in detecting the presence of Helicobacter pylori in the stool due to the fact that many organisms are also present in
the stool sample which can also lead to ulcer originating from infection from the intestine.
Keywords; Ulcer, Helicobacter pylori, Non-invasive method, Antigen test kits, Antibacterial activity.
1.INTRODUCTION
Helicobacter pylori are one of the most common infections in humans, with an estimated 50% of the world’s population
being infected (Torres et al, 2000). This organism has been implicated in the pathogenesis of active and chronic gastric,
peptic ulcer and gastric cancer. Childhood has been identified as the critical time for acquisition of H. pylori
infection(Malaty et al.,2002 and Rowland et al., 2006).The exact route of transmission remains unclear but the
infection clusters in families and familial spread is thought to be the major mode of transmissions ,both in developed
countries(Malaty et al.,2002).
Gastric and ulcer peptic disease is a common disease in the community, especially in children. Considering the close
relationship between peptic ulcer and gastritis caused by Helicobacter pylori, the accurate and early diagnosis of
infection by this microorganism is very important so as to provide prompt and convenient treatment to the affected
children (Torres et al., 2000).
Helicobacter pylori is a helical shaped, gram negative , microaerophilic bacterium(2-4m long with diameter of
0.5pm)that infects the stomach and duodenum of humans(Sasaki et al.,1999); and has been recognized as a class I
carcinogen(Sasaki et al., 1999). They were initially noticed as clinically important by Robin Warren and Barry
Marshall from the human gastric mucosa in 1982; and the demonstration of its involvement in gastro duodenal
pathologies has radically changed peoples’ perception of these diseases (Marshall and Warren, 1983).
Helicobacter pylori transmission remains poorly understood, however it is believed to spread from person to person
through person-person by oral-to-oral, gastro-oral and fecal –to-oral routes(Frenck and Clemens,2003)). Such
observations suggest that these infectious may be associated with low socioeconomic status and overcrowded living
conditions (Deplort et al.,2006).Studies have documented a higher prevalence in Africa(Asrat et al., 2004;Ndip et al.,
2004;Ndip et al.,2008).
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Two categories of diagnostic methods for H. pylori infection are defined; invasive tests to detect the microorganisms in
biopsies sample of the gastric mucosa obtained through endoscopy, and non invasive test which obviate the need for
endoscopy, the ideal diagnostic test for H. pylori infection would be non-invasive, reliable, low-cost and easily
accessible(Clemens et al.,1996).This was used in this experiment, diagnosis of Helicobacter pylori infection generally
relies on a combination of invasive and non-invasive methods Helicobacter pylori. Culture of H. pylori is considered the
gold standard for the diagnosis of the infection, but gastro copy is more complicated in children than in adults
(Clemens et al., 1996).The non invasive techniques include urea breath tests, serological assays and antigen-in-stool
tests and are very useful for detecting Helicobacter pylori infection, especially in children. These techniques are widely
used and recommended as routine diagnostic tools in high-income countries but are not yet established in most
developing countries. This therefore led to the evaluation of prevalence of the organism among sex or gender and in
children as well. Antibiotics therapy is used to eradicate Helicobacter pylori infection. This was shown in the result
obtained from the sensitivity test carried out. The ideal temperature used was 370C rather than the higher temperature
of 420C. More than 30 helicobacter species have been isolated, some infecting occasionally also humans e.g. H.
heilmannii, H. fnelliae and H. pullorum but the primary hosts for the non H pylori species are animals such as dogs(H.
cani),cats(H. fells),pigs(H. suis)and rats(H. hepaticus, H. rodentum, H. bills).
2.AIM AND OBJECTIVES
AIM
To determine Helicobacter pylori infection among patients diagnosed with ulcer using stool and serum antigen test kits.
OBJECTIVES
To evaluate the prevalence of Helicobacter pylori among sex or gender
To evaluate the prevalence of Helicobacter pylori in three different locations
To assess the reliability of the stool and Serology antigen test kits
SIGNIFICANCE OF THE STUDY
It is usually very difficult to treat ulcer, this study will help to provide effective ways in the prevention and treatment of
ulcer to the medical personnel’s and public environment. It will also help the medical personnel’s in knowing which of
the test is more accurate and reliable between stool and serum tests in detecting Helicobacter pylori. The public should
also be more cautious of the importance of practicing good hygiene and having a good sanitation of the environment.
MATERIALS AND METHOD
STUDY AREA
The sample were collected and the work was conducted at the Department of Medical Microbiology ,University College
Hospital Ibadan, General Hospital Ijebu-ode, Ogun State and general Hospital, Oke-Odo, Lagos.
STUDY POPULATION
The study population was drawn from ulcer patients from three different location, comprising 50 patients from Lagos,
40 patients from Ibadan and 50 patients from Ogun(male and female) making the total of 140 over a period of August
2011-November 2011 all patients had clinical evidence of the infection as determined by the treating doctors.
PREPARATION OF MEDIA
All dehydrated media were prepared according to Manufacturers instruction. They were mixed with distilled water and
dissolved by gentle heat to boil. The media were sterilized in an autoclave at 1210C for 15minutes.The sterile media
were dispensed into sterilized Petri dishes and were allowed to cool.
SAMPLE COLLECTION
Stool sample and blood samples were collected from ulcer patients (male and female) in three hospitals from three
locations (Lagos, Ibadan and Ijebu-ode, Ogun State).Stool samples were collected in a universal bottle while the blood
samples were collected in a sodium heparin bottle.
METHOD: Detection of Helicobacter pylori Antigens using
(1.) Stool antigen test; this was carried out in two forms namely;
(a) Solid fecal samples:-the collector applicator was used to stab the fecal sample at three different sites to collect
appropriately 50mg of faeces(equivalent to ¼ oral pea).This is then mix in an extraction buffer found in the stool
antigen test kit pack(H. Pylori Antigen test device (Acon San Diego, CA, USA)
(b) Liquid faecal samples-two drops of the faecal sample were transferred (appropriately 80ml) into the specimen
collection tube containing the extraction buffer. This is then shaken together and left for about 2minutes,the test kits is
then removed from its pouch and about 2-3 drops of the mixed stool and buffer solution is transferred into the specimen
well of the test kit and left for 10 minutes.
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International Journal of Application or Innovation in Engineering & Management (IJAIEM)
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Volume 4, Issue 7, July 2015
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3.RESULTS
Positive; two distinct colored lines appear, one colored line should be in the control line region ( C ) and another
colored line would be in the test region (T ).
Negative; one colored line would appear in the control region ( C ),no line in the test region (T )
Invalid; control line fails to appear, it could be as a result of insufficient specimen or wrong procedure techniques.
(2.) Blood antigen test; after collection of the blood sample from the ulcer patients, the blood was immediately
transferred into the heparinized bottle. The blood was then centrifuged so that the serum can be separated from the
blood. The test kit (H. pylori Antigen test device Acon San Diego, CA,USA) was then removed from its pouch and
placed on a clean and sterile level surface, after which 3-4 drops of serum was dropped on the specimen well on the test
kit using a dropper. The test result is then read after 10 minutes.
RESULT
Positive; two distinct colored lines appear, one colored line in the control line region ( C) and another colored line
would be in the test region ( T ).
Negative; one colored line would appear in the control region (C) no line in the test region (T)
Invalid; control line fails to appear, it could be as a result of insufficient specimen or wrong procedure techniques.
ISOLATION PROCEDURE FOR OTHER MICROORGANISMS IN THE STOOL SAMPLES THAT TESTED
NEGATIVE TO Helicobacter pylori
After the preparation o the agars according to the manufacture directions, they were poured into sterile Petri dishes and
left for the agars to jell then wire loop is use to pick small portion of the stool sample from three (3) sites, a well is
made on the media then the wire loop is flamed and used to make different streaked segments on the agar surface
starting from where the well was made. The plates were then incubated at 370C aerobically for 24 hours after which
they were examined for identification and characterization (MacConkey and Salmonella/Shigella agars).
SUBCULTURE, CHARACTERIZATION AND IDENTIFICATION OF ORGANISMS ISOLATED FROM
STOOL SAMPLES.
Isolates obtained from the MacConkey and Salmonella/Shigella agar were sub cultured on Nutrient agar to obtain pure
isolates of the organism.
The pure cultures of bacteria isolated were characterized using microscopic, physiological and biochemical tests. The
colonies were observed physically on media plate to observe characteristics such as color, shape elevation while cell
morphologies were observed microscopically after gram staining. Various biochemical tests were carried out on the
bacteria isolates for possible identification.
SENSITIVITY TEST
Organisms were picked with a loop and transferred into a tube of Nutrient broth and allow to grow overnight. The
plates containing Nutrient agar were then inoculated with the Nutrient broth containing each organism. Excess
inoculums were removed, rotating the plate through an angle of 600 after each application so that there would be an
evenly spread on the plate and allowed to dry in air but covered to prevent atmospheric contamination (room
temperature). The concentration of the antibiotic discs used are Streptomycin(30µg),Septrin_Cotrimoxazole(30µg),
Chloramphenicol(30µg),
Sparfloxacin(10µg),Ciprofloxacin(10µg),
Amoxicillin(30µg),
Augmentin(30µg),
Gentamicin(10µg), Sparfloxacin(30µg), Tarivid(10µg), the antibiotic discs were then placed on the inoculated plates
using a sterile forceps, then the plates were incubated aerobically for 24hrs at 370C after which they were examined for
zones of inhibition.
RESULTS
Out of 140 stool and blood samples collected from the patients from the three different location, 42(30%) were positive
for the prevalence of Helicobacter pylori in stool sample while 32(22.8%) were positive for the prevalence of
Helicobacter pylori in blood sample.
Table 1, the stool antigen test kit was positive in 42/140(30%) and negative in 98/140(70%) from the three locations,
the serological tests of 140 samples were positive for 32/140(22.8%) and negative for 108/140(77.1%) from the three
major locations. The results also shows that male (71.43%) are more marked for Helicobacter pylori compare to the
females (28.57%) and also more prevalent in Ogun (50%) compared to Lagos (21%) and Ibadan (29%) in figure 1.
Diagnostic accuracy of the two used test kit stool and serum antigen test kit, from the results stool antigen test kit is
more accurate compared to the serum antigen test kit which gave negative results to some of the stool samples that
tested positive from same patient in figure 2 below.
Antimicrobial sensitivity was carried out on the bacteria isolated from the 51 stool samples that showed growth on the
two media used using a gram negative disc (MAXI Nigeria) and their zone of inhibition (mm) was measured. Table 8
shows the antimicrobial sensitivity and zone of inhibition of the bacteria in the three states respectively.
Volume 4, Issue 7, July 2015
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International Journal of Application or Innovation in Engineering & Management (IJAIEM)
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Volume 4, Issue 7, July 2015
Sex
Male
Female
Total
ISSN 2319 - 4847
Table 1: Gender distribution positive to Helicobacter pylori from the three locations.
Positive to H. pylori in Positive to H. pylori in Positive to H. pylori in
Lagos state
Ogun state
Ibadan
6
15
9
3
6
3
9
21
12
Table 2; Results of Helicobacter pylori using stool and serology antigen tests from ulcer patient in three locations in
Nigeria.
Locations
Blood
Stool
Lagos
4
9
Ogun
19
21
Ibadan
9
12
Total
32
42
Figure 1: shows the distribution of H. pylori from three locations based on sex
Figure 2: shows the performance between stool and serum antigen test.
Volume 4, Issue 7, July 2015
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Volume 4, Issue 7, July 2015
ISSN 2319 - 4847
16
18
00
00
00
18
00
00
16
00
16
00
15
15
12
00
00
14
00
00
00
14
00
00
00
00
11
00
00
00
10
18
10
00
00
00
14
00
00
13
00
17
13
00
00
00
11
00
00
13
18
17
18
15
10
18
14
15
15
13
15
00
00
00
00
00
00
11
00
00
00
00
Septrin
Volume 4, Issue 7, July 2015
Ciprofloxacin
Augmentin
Amoxallin
Ciprofloxacin
Amoxallin
Augmentin
Augmentin
Amoxallin
Ciprofloxacin
Sparfloxacin
Chloramphenicol
Septrin
Chloramphenicol
Septrin
00
15
Antibiotics
disc(µg)
12
Streptomycin
17
Chloramphenicol
00
Shigella spp
18
Escherichia colii
00
Klebsiella spp
00
Salmonella spp
Shigella spp
14
Antibiotics
disc(µg)
Escherichia colii
15
Streptomycin
Klebsiella spp
Shigella spp
Salmonella spp
Escherichia colii
11
Antibiotics
disc(µg)
Klebsiella spp
14
Streptomycin
Salmonella spp
Table 3: Sensitivity test of the bacteria isolated from the stool samples obtained from Lagos, Ibadan and Ogun that
tested negative for Helicobacter pylori. Zones of inhibition (mm)
Lagos
Ibadan
Ogun
Page 62
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Volume 4, Issue 7, July 2015
00
15
18
00
00
18
17
17
13
5
00
00
10
00
00
18
13
00
15
15
00
00
00
14
00
00
16
Pefloxacin
Pefloxacin
Tarivid
10
Gentamicin
00
10
Pefloxacin
14
14
Tarivid
17
Gentamicin
14
Tarivid
10
Gentamicin
17
ISSN 2319 - 4847
4.DISCUSSION
In this research work, Helicobacter pylori infection leads to immediate development of persistent gastritis, colonization
of the stomach by Helicobacter pylori is almost always accompanied by clinical and histological signs of chronic
gastritis associated with both local and systematic immune response.(Hopkins et al., 1990)
On sex difference, Helicobacter pylori has been found to be prevalent in males compared to females due to some factors
such as smoking, hard labour, there was statistical difference(p<0.05) between male and female patients studied this
result agreed with the result of Ukaji et al(2011),in which prevalence of Helicobacter pylori was also predominant in
male sex. There was significant difference in Helicobacter pylori prevalence among the three different locations in
Nigeria, Ogun had the highest infection, followed by Ibadan and lowest were seeing in Lagos this as a result as the
influence of socio-economic status. This is consistent with result of previous studies conducted in Nigeria, which have
consistently shown a high prevalence of Helicobacter pylori in South Western part of Nigeria with the use of biopsy
method (Baako et al., 1996).
In this study carried out in the three locations, the percentage of positives obtained for stool antigen test was high when
compared to serum antigen test. This indicates the monoclonal antibody based Helicobacter pylori antigen stool test is
more reliable compared to serum antigen test, this is in accordance with previous studies of this test with reports of
sensitivity and specificity in the range of 88.5%-98% and 93.8%-99% respectively (Andrews et al., 2003).
This is also in keeping with the result obtained in England where they compared three stool antigen kits, which the
stool antigen test kit for the detection of Helicobacter pylori has a high sensitivity and specificity (Sykora et al.,
2003).The similarity in the result might be attributed to the condition of the patients and the stool antigen kits. The
stool and serology test were found to be equally acceptable to patients with ulcer for detecting ulcer. The stool antigen
test showed a good performance for the study test; however the performance of the stool antigen test kit is dependent to
the prevalence of the infection and therefore high in population with high prevalence of Helicobacter pylori infection
such as in Ogun State. If the prevalence of Helicobacter pylori infection in a location is lower, the performance of the
test will be accordingly lower, i.e. the positive predictive value will decrease.
Antibiotic sensitivity was carried out on the bacteria organisms present using gram negative disc (MAXI Nigeria),
Streptomycin was the most potent of all with zones o inhibition ranging from 16mm-18mm,It was effective against all
the test organisms while antibiotic like Augmentin did not inhibit any of the test organism while Septrin was only
effective against Escherichia colii the rest were effective against Salmonella spp and Shigella spp.
The bacterium makes up approximately 10% of the intestinal microorganisms of human and were widespread used as
indicator organism. These bacteria is assumed to have been the cause of the ulcer disease or other cause such as the use
of NASID such as aspirins or socio-economic of the population. From the past works on this study, none of the journals
has been able to go ahead to culture stool samples that tested negative to Helicobacter pylori using the stool antigen test
kits which were carried out in this study.
5.CONCLUSION
Result of this study showed that there was a significant difference in Helicobacter pylori infection among the male and
female sex which was prevalent in that of male (30(71.43%) than female (12(28.57%) patients and that of the locations
also Ogun State having the highest infection followed by Ibadan and lowest were seen in Lagos due to the socioeconomic status of the locations. From the statistical analysis, it showed that there is not much difference in the
performance of the stool (42%) and serum (32%) antigen test using the serology antigen test kit from -Acon San Diego,
CA, USA.
Volume 4, Issue 7, July 2015
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Volume 4, Issue 7, July 2015
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RECOMMENDATION
Helicobacter pylori is an etiologic factor for ulcer, therefore the medical community should seriously consider the merit
of early screening of patients with Helicobacter pylori and precautions should be taken against the bacterium and other
factors such as smoking, drinking of excess alcohol, use of NSAID drugs and good sanitation.
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[17.] Ndip, R.N. (2004).Helicobacter pylori antigens in the feaces of asymptomatic children in the Buea and Limbe
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MacMillan, M and Weaver T.L(2008).Helicobacter pylori isolates recovered from gastric biopsies of patients with
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[26.] Scott, D.R.,(2002).Mechanisms of acid resistance due to the Urease system of Helicobacter pylori. gastroenterol
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APPENDIX
Statistical analysis; data were analyzed using ANOVA table and Chi-square, P values less than 0.05 were considered
significant.
Table for figure 1: gender distribution positive to Helicobacter pylori from the three locations.
Sex
Positive to H pylori in Positive to H. pylori in Positive to H. pylori
Lagos State
Ogun State
in Ibadan
Male
6
15
9
Female
3
6
3
Total
9
21
12
Table for figure 2: performance between stool and blood serology antigen test.
State
Blood
Stool
Lagos
4
9
Ibadan
9
12
Ogun
19
21
Total
32
42
Data analysis and interpretation
Table 3: Summary of result of Helicobacter pylori using stool and blood antigen test from stool and blood samples
collected from ulcer patients in Lagos, Ibadan and Ijebu-ode.
Locations
Antigen test
Blood
Stool
Total
Lagos
4
9
13
Ibadan
9
12
21
Ogun
19
21
40
Total
32
42
74
Table 4: ANOVA TABLE BASED ON THE ANALYSIS USING RCBD
Source of variation D.F
Sum of Squares
Mean Square
F
Antigen test
1
16.66
16.66
14.24
Location
2
192.33
92.165
78.77
Error
2
2.34
1.17
Total
5
211.33
109.995
Sex
Locations
Male
Lagos
Ogun
Ibadan
Female
Lagos
Ogun
Ibadan
Total
Hence, X2calculated =Σ (O-E) 2/E =0.1522
Hypothesis testing
Test 1; Antigen Test
Volume 4, Issue 7, July 2015
Table 5: Chi-square analysis table
Observed
Expected
O-E
(O)
(E)
6
6.4
-0.4
15
15.0
0
9
8.6
0.4
3
2.6
0.4
6
6.0
0
3
3.4
-0.4
42
42
0.0
(O-E)2
(O-E)2 /E
0.16
0
0.16
0.16
0
0.16
0.64
0.025
0
0.0186
0.0615
0
0.0471
0.1522
Page 65
International Journal of Application or Innovation in Engineering & Management (IJAIEM)
Web Site: www.ijaiem.org Email: editor@ijaiem.org
Volume 4, Issue 7, July 2015
ISSN 2319 - 4847
H0: There is no significant difference between the antigens tests used.
H1: There is significant difference between the antigens tests used.
The critical value of F is calculation as:
F 1; 2(5%) =18.5
Decision rule; Reject the null hypothesis (H0) and accept the alternative hypothesis (H1) when
F calculated is greater than F tabulated value.
Conclusion; thus, since F calculated value 14.24 is less than F tabulated value i.e.18.5 (14.24<18.5): then we accept the null
hypothesis (Ho) and conclude that the experiment failed to show any significant difference among the two antigen test
used. Therefore, the Blood and Stool antigen test are both effective.
Test 2(Location)
Ho: There is no significant difference between the locations.
H1: There is significant difference between the locations.
The critical value of F is calculated as:
F1;2(5%)=19.0
Decision rule: Reject the null hypothesis (Ho) and accept the alternative hypothesis (H1) when Fcalculated is greater than
Fcalculated value.
Conclusion; thus, since Fcalculated value i.e.78.77 is greater than Ftabulated value i.e. 19.0(78.77>19.0): we reject the null
hypothesis (Ho) and conclude that there is significant difference among the three locations. Therefore, this show that
there are some factors affecting the patients in each location, factor such as; Age, overcrowding, income,
socioeconomic of the groups etc.
The degree of freedom for the Chi-square distribution is
V= (h-1)(k-1)=(2-1)(3-1)=2
Therefore,
X2tabulated =X22, 0.05
Hypothesis testing
Ho; the positive to Helicobacter pylori in each state are independent of sex.
H1: The positive to Helicobacter pylori in each state are dependent of sex.
Decision rule; Reject the null hypothesis (Ho) and accept the alternative hypothesis (H1) when X2calculated is greater than
X2tabulated value.
Conclusion; Thus, since X2calculated value is less than X2tabulated value i.e. 0.1522<5.991, we cannot reject the null
hypothesis (H0) so, we accept the H0 and conclude that the positive to Helicobacter pylori in each state is independent
of sex, and also from the observed table, in the states, for instance, a significantly lower prevalence was found in Lagos
(21%) compared to Ibadan (29%) and Ogun (50%) with higher prevalence of Helicobacter pylori. This dissimilarity
was interpreted to reflect the different socioeconomic backgrounds of the groups. The infection is also associated with
low SES within the states (Graham et al.1991).
Descriptive statistics
N
Mean
Std Deviation
Minimum
Maximum
Observed
42
2.93
1.504
1
6
Volume 4, Issue 7, July 2015
Page 66