Download Activity test of Commiphora myrrh to Helicobacter pylori compared

Activity test of Commiphora myrrh to Helicobacter pylori
compared with commonly used
Antimicrobial Agents
Thesis Submitted by
Huda Mohamed El Mahdi Salih
B. Sc 2000 Sudan university
For degree of master of microbiology
Under supervision of
Prof. Elshiekh Ali Elebiad
Department of microbaiology
Faculty of pharmacy
University of Khartoum
2008
‫ﻗﺎل ﺗﻌﺎﻟﻰ ‪:‬‬
‫ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ‬
‫ﻼ(‬
‫ﻻ َﻗﻠِﻴ ً‬
‫) َوﻣَﺎ أُوﺗِﻴﺘُﻢ ﻣﱢﻦ ا ْﻟ ِﻌ ْﻠ ِﻢ ِإ ﱠ‬
‫ﺻﺪق اﷲ اﻟﻌﻈﻴﻢ‬
‫اﻹﺳﺮاء اﻵﻳﺔ) ‪( 85‬‬
‫‪I‬‬
Dedication
To Soul of my
Father and grand father
To my mother and my Family
To my sister Hwaida who spared no effort to help me
To my husband & my lovely Kids
Hosham & Aymn
To all whom I love
II
Acknowledgment
I wish to express my special appreciation and sincere gratitude to Prof. Elshei kh Ali Elobaid for close
supervision and continuous moral support.
My thanks also to my co –supervisor Dr. Elamin Ibrahim Elnema for his kind help .
Also special thanks to Dr. Hekma Yousif Mohamed Elzin for her continuous support .
I also extend my warmest thanks to my colleagues at N.H.L for their continuous support
Also I would like to extended my thanks to the staff of endoscopy department and staff of laboratory
of Ibn Sinna Hospital and specially to Abd Elrazig Hammad Suleiman
I would like to thank
Dr. Amira Mahjoub Mycoplasma Dept. Central Veterinary Research laboratories Center Soba
Mr. Awad Elsead Abd Elgbar Mycoplasma Dept. Central Veterinary Research laboratories Center
Soba
N.H.l.. bacteriology department members and growth media unit
The epidemiology department members manly Mohamed abd Elrahman and Vaccine department
members at N.H.L
Mr Ali M. Nor Eldein. Laboratory Administration Khartoum State
Dr. Eldoma H. Adil N.H.LVirology department
Miss Kawthar Abdelgaleil Mohamed Salih N.H.L. Immunology & Allergy department
Sarah Elsarag & Salah Elrwa my friend & colleague whom I sheered the hardest
day
Mr. Malik Abd Allah N.H.L. and Mrs. Suzan Fathi
I am extremely grateful for support & advice and cooperation from my lovely famil
III
Abbreviations
CagA
CD
IgG
IgM
IL8
BHI
CFU
IgA
HpSA
H. pylori
H. bizzozeronii
STAT
LPS
PCR
RNA
UBT
VacA
NF.B
PUD
PPI
ELISA
ZES
MALT
NSAID
Am
Tet
Cla
Met
RBC
Clo
Pu
DU
GU
cytotoxin associated gene A
cluster of differentiation
immunoglobulin G
immunoglobulin M
interleukin8
Brain heart infusion
colony forming units
immunoglobulin A
Helicobacter pylori stool antgen
Helicobacter pylori
Helicobacter bizzozeronii
signal transducer and activator of transcription
lipopolysaccharide
polymerase chain reaction
ribonucleic acid
Urea breath test
Vacuolating cytotoxin A
Nuclear factor B
Peptic ulcer disease
proton pump inhibitor
enzyme linked immunosorbant assay
Zollinger-Ellison syndrome
mucosa associated lymphatic tissue
Non steroidal anti-inflammatory
Amoxicilin
tetracycline
Clarithromicin
Metronidazole
ranitidine bismuth citrate
Campylobacter-like organism test
peptic ulcer
duodenum ulcer
gastric ulcer
IV
Abstract
The present study aimed to isolate Helicobacter pylori and conduct the susceptibility
tests to Commiphora myrrh in comparison with widely used antibiotics. The study
samples were collected from Sudanese patients who were suffering from peptic disease
during the period September 2006 to November 2007 in Ibn Sina T. Hospital.
The study population was 52 females and 48 males their ages ranged between 19 and 70
years. It was observed that high prevalence of Helicobacter pylori infection in old ages .
The study included 32 manual worker, 31 house wives and 15 students , 6 farmers and
16 other jobs (Doctors ,Employers ,Engineers and Teachers). Helicobacter pylori was
isolated in sixty of the total of one hundred specimen collected. The sensitivity tests
were done against Amoxicillin, tetracycline ,Metronidazole and Clarithromycin by disk
diffusion method and Commiphora myrrh extraction by agar dilution in four dilution
1/20,1/40,1//80 and 1/160. It was found that 71% of isolates were sensitive to
Amoxicillin, 61.7%Tetracycline ,38.3% to Metronedazole and35% to Clarithromicin ,In
Commiphora myrrh dilution one (1/20)which highist sensitivity rate 100% . Dilution
two(1/40) 90% .50% for dilution three(1/80). And 20 % for dilution four (1/160) . That
means that Amoxicillin is the best choice and the MIC of Commiphora myrrh extraction
is 1/20 and Commiphora myrrh may be examined as an alternative treatment against
Helicobacter pylori .
V
‫ﺒﺴﻡ ﺍﷲ ﺍﻟﺭﺤﻤﻥ ﺍﻟﺭﺤﻴﻡ‬
‫ﻤﺴﺘﺨﻠﺹ ﺍﻷﻁﺭﻭﺤﺔ‬
‫ﻫﺩﻓﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺍﻟﺤﺎﻟﻴﺔ ﻟﻌﺯل ﻤﻴﻜﺭﻭﺏ) ﺍﻟﻬﻴﻠﻜﻭﺒﺎﻜﺘﺭ ﺒﻴﻠﻭﺭﻯ( ﻭﺩﺭﺍﺴﺔ ﺩﺭﺠﺔ ﺤﺴﺎﺴﻴﺘﻬﺎ ) ﻟﻤﺭ ﻫﺎﺭﺒـل( ﻤـﺎ‬
‫ﻴﻌﺭﻑ ﻓﻰ ﺍﻟﺴﻭﺩﺍﻥ ﺒﺎﻟﻤﺭ ﺍﻟﺤﺠﺎﺯﻯ ﻤﻘﺎﺭﻨﺔ ﺒﺎﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﺤﻴﻭﻴﺔ ﺍﻻﻜﺜﺭ ﺍﺴﺘﺨﺩﺍﻤﺎ ﻓﻰ ﺍﻟﻌﺎﻟﻡ‪.‬‬
‫ﺃﺠﺭﻴﺕ ﺍﻟﺩﺭﺍﺴﺔ ﻋﻠﻰ ﻋﻴﻨﺎﺕ ﺍﻨﺴﺠﺔ ﻤﻥ ﻤﺭﻀﻰ ﺴﻭﺩﺍﻨﻴﻴﻥ ﻴﻌﺎﻨﻭﻥ ﻤﻥ ﺍﻟﻘﺭﺤﺔ ﺍﻟﻤﻌﺩﻴﺔ ﺨـﻼل ﺍﻟﻔﺘـﺭﺓ ﻤـﻥ‬
‫ﺴﺒﺘﻤﺒﺭ ‪ 2006‬ﺍﻟﻰ ﻨﻭﻓﻤﺒﺭ‪ 2007‬ﺒﻤﺴﺘﺸﻔﻰ ﺍﺒﻥ ﺴﻴﻨﺎ ﻗﺴﻡ ﺍﻟﻤﻨﺎﻅﻴﺭ ‪.‬‬
‫ﺸﻤﻠﺕ ﺍﻟﺩﺭﺍﺴﺔ ‪ 52‬ﻤﻥ ﺍﻻﻨﺎﺙ ﻭ‪ 48‬ﻤﻥ ﺍﻟﺭﺠﺎل ﺘﺘﺭﺍﻭﺡ ﺍﻋﻤﺎﺭﻫﻡ ﺒﻴﻥ ‪ 19‬ﺍﻟﻰ ‪ 70‬ﺴﻨﺔ ﻭﻭﺠـﺩﺕ ﺍﻻﺼـﺎﺒﺔ‬
‫ﺒﺎﻟﻬﻴﻠﻭﻜﻭﺒﺎﻜﺘﺭ ﺒﻴﻠﻭﺭﻯ ﺍﻜﺜﺭ ﺸﻴﻭﻋﺎ ﻓﻰ ﺍﻻﻋﻤﺎﺭ ﺍﻻﻜﺒﺭ ‪.‬‬
‫ﻭﺸﻤﻠﺕ ﺍﻟﺩﺭﺍﺴﺔ ‪ 32‬ﻤﻥ ﺍﺼﺤﺎﺏ ﺍﻻﻋﻤﺎل ﺍﻟﺤﺭﺓ ﻭ‪ 31‬ﻤﻥ ﺭﺒﺎﺕ ﺍﻟﺒﻴﻭﺕ ‪ 15‬ﻤﻥ ﺍﻟﺘﻼﻤﻴﺫ ﻭ‪ 6‬ﻤﺯﺍﺭﻋﻴﻥ ﻭ‪16‬‬
‫ﻤﻥ ﺍﻟﻤﻬﻥ ﺍﻻﺨﺭﻯ ) ﺍﻁﺒﺎﺀ ﻤﻬﻨﺩﺴﻴﻥ ﻭﻤﺤﺎﻤﻴﻴﻥ ﻭ ﻤﻌﻠﻤﻴﻥ (‬
‫ﻋﺯﻟﺕ ﺒﻜﺘﺭﻴﺎ ﺍﻟﻬﻠﻜﻭﺒﺎﻜﺘﺭ ﺒﻴﻠﻭﺭﻯ ﻤﻥ ﺴﺘﻴﻥ ﻋﻴﻨﺔ ﻤﻥ ﻤﺠﻤﻭﻉ ﻤﺎﺌﺔ ﻭﺍﺠﺭﻱ ﻟﻬﺎ ﺍﺨﺘﺒﺎﺭ ﺍﻟﺤﺴﺎﺴﻴﺔ ﻟﻠﻤﻀﺎﺩﺍﺕ‬
‫ﺍﻟﺤﻴﻭﻴﺔ )ﺍﻤﻭﻜﺴﺴﻠﻴﻥ ﻭﺘﺘﺭﺍﺴﻴﻜﻠﻴﻥ ﻭﻜﻼﺭﻴﺜﺭﻭﻤﺎﻴﺴﻥ ﻭﻤﻴﺘﺭﻭﻨﻴﺩﺍﺯﻭل (ﺒﻁﺭﻴﻘﺔ ﺍﻨﺘﺸﺎﺭ ﺍﻻﻗﺭﺍﺹ ﺍﻟﻤـﺸﺭﺒﺔ ﻭﻟﻠﻤـﺭ‬
‫ﺍﻟﺤﺠﺎﺯﻯ ﺒﻁﺭﻴﻘﺔ ﺘﺨﻔﻴﻑ ﺍﻻﻗﺎﺭ ﺒﺎﻟﺘﺨﻔﻴﻑ ﺍﻟﻤﻀﺎﻋﻑ ‪20/1‬ﻭ‪ 40/1‬ﻭ‪ 80/1‬ﻭ‪ 160/1‬ﻭﻭﺠﺩ ﺍﻥ ﻨـﺴﺒﺔ ﺍﻟﺘﺤـﺴﺱ‬
‫ﻟﻼﻤﻭﻜﺴﺴﻠﻴﻥ ‪ %71‬ﻭﻟﻠﺘﺘﺭﺍﺴﻴﻜﻠﻴﻥ‪ %61‬ﻭﻟﻠﻤﻴﺘﺭﻭﻨﻴﺩﺍﺯﻭل ‪ %38.3‬ﻭ‪ %35‬ﻟﻠﻜﻼﺭﻴﺜﺭﻭﻤﺎﻴـﺴﻥ ﻭﺒﺎﻟﻨـﺴﺒﺔ ﻟﻠﻤـﺭ‬
‫ﺍﻟﺤﺠﺎﺯﻯ ﺍﻋﻁﻰ ﺘﺤﺴﺱ ﻋﺎﻟﻰ ﺒﻨﺴﺒﺔ ‪ %100‬ﻓﻰ ﺍﻟﺘﺤﻔﻴﻑ ﺍﻻﻭل )‪ (20/1‬ﻭ‪ %90‬ﻟﻠﺘﺤﻔﻴﻑ ﺍﻟﺜﺎﻨﻰ)‪ (40/1‬ﻭ‪%50‬‬
‫ﻟﻠﺘﺨﻔﻴﻑ ﺍﻟﺜﺎﻟﺙ )‪ (80/1‬ﻭﻜﺎﻨﺕ ﻨﺴﺒﺔ ﺍﻟﺘﺤﺴﺱ ﻓﻲ ﺍﻟﺘﺨﻔﻴﻑ ﺍﻟﺭﺍﺒﻊ ﻭﺍﻻﺨﻴﺭ )‪%20 (160/1‬‬
‫ﻫﺫﺍ ﻴﻌﻨﻰ ﺍﻥ ﺍﻻﻤﻭﻜﺴﺴﻠﻴﻥ ﻭﺍﻟﺘﺘﺭﺍﺴﻴﻜﻠﻴﻥ ﻫﻤﺎ ﺍﻟﺨﻴﺎﺭﻴﻥ ﺍﻻﻓﻀﻠﻴﻥ ‪ .‬ﻭﺍﻥ ﺍﻗل ﺘﺭﻜﻴﺯ ﻟﻠﻤﺭ ﺍﻟﺤﺠـﺎﺯﻯ ﺍﻟـﺫﻯ‬
‫ﻴﺜﺒﻁ ﻨﻤﻭ ﺍﻟﺒﻜﺘﺭﻴﺎ ﻫﻭ ‪ ،20/1‬ﻜﻤﺎ ﺍﻨﻪ ﻴﻌﻨﻰ ﺍﻥ ﺍﻟﻤﺭ ﺍﻟﺤﺠﺎﺯﻯ ﻴﻤﻜﻥ ﺍﻥ ﻴﺨﺘﺒﺭ ﻜﻌﻼﺝ ﺒﺩﻴل ﻀـﺩ ) ﺍﻟﻬﻠﻜﻭﺒـﺎﻜﺘﺭ‬
‫ﺒﻴﻠﻭﺭﻱ(‪.‬‬
‫‪VI‬‬
Table of content
Title
Page No.
Verse
I
Dedication
II
Acknowledgment
III
Abbreviations
IV
Abstract in English
V
Abstract in Arabic
VI
Chapter One
Introduction and Literature review
Peptic Ulcer
1
1
Causes of peptic ulcer
2
Symptoms of peptic ulcer
4
Other symptoms
4
Emergency Symptoms
4
Treatment of peptic ulcer :
5
Helicobacter
5
Characteristic
6
Habitat
6
Species
6
Antigenic structures
7
culture and colony morphology
7
Pathogenesis
8
Diseases
9
Diagnosing
10
VII
Histology
10
Culture
11
Rapid urease test
11
Urease breath test
12
Serology
12
Stool antigen test
13
Near patient test
14
Salivary and urine antibody
14
Molecular test
14
String test
15
Immunity
15
Transmission
15
Epidemiology
16
Treatment
16
First-line treatment
16
Second-line therapies
18
Resistance
18
Medical plants
19
The activity of medical plants
19
Commiphora myrrh
20
Active Compounds
22
Objective of the study
23
Chapter Two
Materials and methods
24
Study approach
24
Study design
24
VIII
Study area
24
Study variables
24
Study population
24
Selection criteria
24
Ethical clearance
24
Material
24
Methods
26
Collection of the samples
26
Rapid urease test
26
Culture
26
Identification of the isolate:
27
Colonial morphology
27
Gram stain
27
Urease test
27
Catalase test
28
Oxidase test
28
Sensitivity test
28
Inoculums’ preparation
28
Disk diffusion methods
29
Disc diffusion method of antimicrobial
29
Agar dilution method
29
data analysis
30
Chapter Three
Result
31
Demographic features of study population
31
Gender And Age
31
IX
Residence distribution
31
Occupation distribution
31
Endoscopy result
31
Rapid urease test result
31
Culture result
32
Sensitivity test result:
32
Antibiotic desk result
32
Plant antimicrobial result
32
Chapter Four
Discussion
53
Chapter Five
Conclusion
57
Recommendation
58
References
60
X
List Of table
Title
No
Table (1) Age distribution
34
Table (2)Rapid urease test
38
Table (3)Culture distribution
39
Table (4)Culture *Rapid urease test Cross
tabulation
40
Table (5)Gender * Culture Cross tabulation
Table (6)Occupation * Culture Cross tabulation
Table (7)Residence * Culture Cross tabulation
41
42
43
List Of Fig
Title
No
33
Fig (1) Gender distribution
Fig (2 Residence distribution
Fig (3) Occupation distribution
Fig (4 Endoscopy result
Fig (5 Culture * Endoscopy Result Cross tabulation
Fig (6 Clarithromycin sensitivity test
Fig (7 Metronidazole sensitivity test
Fig (8 Amoxicillin sensitivity test
Fig (9)Tetracycline sensitivity test
Fig (10) Commiphora myrrh sensitivity test (dil 1
Fig (11) Commiphora myrrh sensitivity test (dil 2)
Fig (12) Commiphora myrrh sensitivity test (dil 3)
Fig (13) Commiphora myrrh sensitivity test (dil 4
XI
35
36
37
44
45
46
47
48
49
50
51
52
Introduction and Literature review
1.1 Peptic Ulcer:
A peptic ulcer is a sore on lining 0f the stomach or duodenum which is the beginning
of small intestine .It refer to an ulcer in the lower esophagus ,stomach, duodenum in
jejunum after surgical anastonsis to stomach or rarely in the ileum adjacent to
meckel’s divverticulum ,in acid and pepsin play major pathogenic role(1,2 ). An ulcer
in the lining of the stomach is called a gastric ulcer and the ulcer on upper part of the
small intestine or duodenum called duodenal ulcer. In the US, duodenal ulcers are three
times more common than gastric ulcers.
The lining of the stomach is a layer of special cells, chemicals and mucous that
prevents the stomach from being damaged by its own acid and digestive enzyme. If
there is a break or ulcer in the lining, the tissue under the lining can be damaged by the
enzymes and corrosive acid . If the ulcer is small ,there may be few symptoms and the
wound can heal on its own .If the ulcer is deep ,it can cause serious pain or bleeding
and may eat completely through the stomach or duodenum wall.
The ulcers are common world wide , it may affect up to10-15 % of the population at is
variable but it is very common in Scotland and northern England more than in southern
England ,is also common in India (3,4) and common in Africa especially in the west,
and usually the most affected patient present with complication ,incidence of P.U.D is
declining in the world especially the complicated type but still the gastric ulcer is
common in the east especially in Japan ( pre-malignant ulcer ) (5),
The prevalence of peptic ulcer is decreasing in many western communities. It sill
affects at some time approximately 10% of all adult males. The male to female ratio for
duodenum ulcer varies from 5:1 to2:1 in different communities while that for gastric
ulcer is 2:1 or less (2). In Sudanese patients peptic ulcer was found in 17% of 2500
upper gastrointestinal endoscopies (6) in other study on 12443 upper gastrointestinal
endoscopies the active duodenal ulcer was 10-8% with seasonal variation seen with
great incidence in January, February and lower incidence in July, August in
comparison with the rest of year .(1,7,8).in other studies in Sudan the prevalence of
P.U.D is about 18-30% so it is a very common disease .It effects predominantly, the
younger age group of which a large number of patient presented with complications
(3,9)
Katelaris et al .,(10) in heir study of dyspepsia ,H. pylori and
peptic ulcer in the randomly selected population in India found that prevalence of
1
active peptic ulcer in 197 male subjects was 6,6% with a further 6,6%of subjects has
definite evidence of scaring or deformity (10) .In Misra et al.(11) study in India on H.
pylori positive asymptomatic healthy subjects were 2,8% (11) .
In Italian (12) study The prevalence was as higher as 22% in a group of 121
individuals with H. pylori infection (12) in asymptomatic healthy blood donors
Peptic ulcer is common in every 10 Americans develops an ulcer of some time in his or
her life. The main cause of an ulcer is bacterial infection but some ulcers were caused
by long- term use of non steroidal anti-inflammatory agents (NSAID) like aspirin and
ibuprofen . In few cases, cancerous tumors in stomach or pancreas can cause ulcer .
1.1.2 causes of peptic ulcer :
The major factor to be examined are H. pylori ,non-steroidal anti-inflammatory drugs
(NSAIDs),smoking stress ,alcohol ,dietary factors ,physical activity and heredity (13) .
Since the Nobel prize-winning demonstration of the role of H. pylori in peptic ulcer
,there has been a widespread assumption that H. pylori is the cause of P.U and so
implicitly of the epidemic (13,14) Baron and Sonnenberg (15) have suggested an
epidemic of a potent strain of H. pylori sweeping the world was the cause of the ulcer
epidemic ,and there are temporal, geographic and theoretical arguments to support this.
The people infected with H. pylori have arlativ risk of 1.6-5.7 (mean 3.3)of developing
PU compared with non-infected people (13,16,17) calculation of the population
attributable risk (ie, the proportion of disease cases that can be attributed to a particular
factor) shows that ,in recent series, H, pylori accounts for only about 50% of peptic
ulcer (18) .
`
Non-steroidal anti-inflammatory drugs (NSAIDs) (13,19,20). Despite a large body of
evidence suggesting a role of non-steroidal anti-inflammatory drug (NSAIDs) in
causing ulcer especially in elderly women (13).
long− term use of non steroidal anti−inflammatory drugs (NSAIDs) is the second most
common cause of ulcers .About 5-10% of duodenal
ulcer disease patients and 30% of gastric ulcer patients do not have evidence of H
.pylori infection .these ulcer presumably due to ASA, NSAIDs or some other agents of
injury(13).About tow of every three patient on long-term NSAIDs have some gastro
duodenal mucosal lesions. most of which were superficial (corrosions, rhanguse,etc)
(3,13)
Non-steroidal anti-inflammatory drugs (NSAIDs) provide effective management of pain
2
and inflammation ,but are associated with the formation of peptic ulcer and increased
risk of peptic ulcer hemorrhage and perforations (serious gastrointestinal events)in the
range o.3%to2.5% per year(20,21,22)
Lifestyle Factors.: Although lifestyle factors (e.g. chronic stress ,dietary factor
,smoking ).
Smoking : there is evidence that smoking increased the risk of peptic ulcer .An OR of
2.2 is typical ,and a 40-year study of male doctors in the U.k has shown a threefold
increase in the risk of death from peptic ulcer in current smokers.(13,29)
Alcohol : there is little evidence of an association between alcohol use and peptic ulcer
.A study of Doll et al found no association in 13 year of follow-up (13,25).
With coffee ,sugar ,salt there is little evidence of a link between them and duodenum
ulcer (26,27,28)
.
Hereditary : A role of hereditary factor in peptic is Cleary delineated by several
Scandinavian studies of large groups of monozygonic and dizygonic twins, reared
together and separately .the evidence indicates that 39% -62% susceptibility to peptic
ulcer is explained by hereditary factor ,the rest by environmental ones (29,30).There is
also a major role for heredity in H. pylori acquisition (63% attributable to genetic
factors), but genetic influences for developing peptic ulcer independent of those for
acquiring H .pylori and the transition of H. pylori positivity to peptic ulcer is due to
environmental factor (31).
Stress : The sparse evidence for a link between stress and peptic ulcer is beset with
methodological problem .A follow-up of 5388people in the first US National Health
and Nutrition Examination Survey (1986-1992)found a clear positive dose –response
curve . Those with the highest level of stress had an adjusted a relative risk of
developing peptic ulcer (31).
- The least common major cause of peptic ulcer disease is the Zollinger−Ellison
syndrome (ZES).\ among patients with peptic ulcers. The mean age at onset is 45 to 50
years, and men are affected more often than women.
ZES should be suspected in patients with ulcers who are not infected with H .pylori
and have no history of NSAID use.
-Rarely, certain conditions may cause ulceration in the stomach or intestine , including
· Radiation treatments.
· Bacterial or viral infections.
· At 50 years old or older .
· Physical injury.
. Burin
3
1.1.3 Symptoms of peptic ulcer :
Pain is the most common symptom. The pain usually:
Is a dull, gnawing ache;
Comes and goes for several days or weeks;
Occurs 2 to 3 hours after a meal;
Occurs in the middle of the night (when the stomach is empty) , and is relieved by
food.
1.1.3.1 Other symptoms:
Other symptoms of peptic ulcer include
Weight loss.
Poor appetite.
Bloating.
Burping.
Nausea.
Vomiting.
Some people experience only very mild symptoms, or none at all.
1.1.3.2 Emergency Symptoms :
Sharp, sudden, persistent stomach pain.
Bloody or black stools.
Bloody vomit or vomit that looks like coffee grounds.
They could be signs of a serious problem, such as:
Perforation-when the ulcer burrows through the stomach or duodenal wall.
Bleeding - when acid or the ulcer breaks a blood vessel.
Obstruction - when the ulcer blocks the path of food trying to leave the stomach .
4
1.1.5 Treatment of peptic ulcer:
Treatment of peptic ulcer is currently in astate of major change .In the past therapy
that consisted of acid-neutralizing or acid- secretary inhibiting drugs was useful to
control the symptoms. (3).
people whose ulcers are caused by NSAIDs or other drugs should stop using these
drugs. Healing will begin almost immediately. Also medications are recommended to
reduce acid damage during
healing .These may include anti-acids to neutralize
gastric acid or medications that decrease the amount of acid produced by the stomach.
1.2 Helicobacter :
In the aearly 1980s, Warren and Marshal, two Australian physicians, reported the
presence of an unidentified gram-negative curved and spiral-shaped bacillus in gastric
epithelial tissue of patient with chronic gastritis .Originally called Campylobacter
pylori this organism was later renamed Helicobacter pylori when the organism was
found to have characteristics that differed from those of true Campylobacters (33)..
In 1982. Warren and Marshal were the first to culture and identify what is now known
as H .pylori and to establish the association between the presence of this organism in
mucosa and occurrence of histological gastritis (34,35). Within a few years of
publication of Warren and Marshal’s work many reports appeared worldwide.
confirming the close association between H .pylori and chronic active gastritis .as well
as with gastric and duodenal ulcer .The historical association between bacteria and
peptic ulcer disease are described by Rathbone and Heatley (1992) (34) The organism
was named Campylobacter pyloridis by Marshal et al (1994).but this was later changed
to the more grammatically correct Campylobacter pylori. Further studies showed that
the organism sufficiently from true Campylobacter to justify the formation of a new
genus Helicobacter.(34,36).It soon became apparent that similar organism colonized
the stomach of a wide variety of animals other than humans .and certain spiral bacteria
colonized intestines of rodents and other animals belonged to Helicobacter
.Helicobacter and Campylobacter, Arcobacter and Wolinella was shown to belong to a
distinct bacterial grouping. RRNA super family VI .based on 16s RNA-DNA
hybridization analysis(37) and the classification was defined by 16s r DNA phylogenic
analysis which placed Helicobacter and these other genera in the epsilon subclass of
class proteobacteria . Although specific fatty acid profile .specific ultra structure.
5
Respiratory quinines. growth requirements and enzyme characteristics were originally
described to distinguish Helicobacters .the use of 16s rDNA analysis is crucial for their
classification as there are no other consistent taxonomic markers to facilitate separation
of Helicobacter from allied genera such as Campylobacter and Arcobacter.
Helicobacter pylori infects about half of the world’s population. was widely accepted
as a cause for the development of gastritis (38,39,40). Today, H. pylori is not only
considered a causative agent of chronic type B gastritis and peptic ulcer disease, but
also an important risk factor for gastric cancer and MALT-lymphoma .
1.2.1 Characteristics:
H. pylori is a curved or spiral shaped Gram negative bacillus with up to three windings
(40). It measures 3-5 µm in length and approximately 0.5 µm in diameter. There are
two to six unipolar sheathed flagella (41), which are essential for the bacterium’s
motility. Under unfavorable conditions H. pylori can transform into a coccoid form,
which probably reflects degenerative changes (42). The microaerophilic bacterium
possesses both oxidase and catalase activities. The complete genome of a H. pylori
strain was sequenced by Tomb et al . (1997) (43 )
1 2.2 .Habitat :
Humans appear to be the only natural host of H. pylori identified to date, although
some studies suggest that animals also may act as a reservoir, as reviewed by Mitchell
(38,44).
The surface of the stomach mucosa is the major habitat of H. pylori and the majority
are located adjacent to surface and pit gastric epithelial cells (34)
1.2.3 Species :
There are more than 20 species ascribed io the Helicobacter genus. Chronic infection
of various host with gastric Helicobacters such a H. pylori, H. felis ,H. mustelae and
H. bizzozeronii has been associated with a spectrum of pathologies such as gastritis,
mucosa-associated lymphoid tissue lymphoma(45,46,47)and in the case of H .pylori ,
peptic ulcer and gastric adenocarcenoma in humans (48,49).Other related bacteria
such as H. hepaticus, H. bilis, H. pullorum and Helicobacter sp flexispira colonize the
enterohepatic niche of animals and humans, and many play role in enterohepatic and
gastroenteritis (50,51).And other spp such as H.acinonychiis ,H.nemestrinae,H
6
.mustelae
(34)
,H.heillmannii,H.canadensis andH.bills ,H.bovis ,H. suis and H. Suncus
2.2.4 Antigenic structures:
Helicobacter have the typical cell wall structure of gram- negative bacteria comprised
of lipopolysaccharide (LPS) attatched to a semi permeable outer membrane
apeptidoglycan layer .and an inner cell membrane. The fatty acid composition of H.
pylori is distinctive and one of the main criteria for Campylobacter’s .It is characterized
by long –chain fatty acid composed predominantly of tetradecanoic (14 :0) and 19carbon cyclopropan (19-0cyc)acid acids with lower amounts of hexadecanoic (16:0)
octadecanoic (18:0) . octadecanoic (18:1) and linoleic (18:2 )acid Phosphatidylethanolamine (79.1%) .lysophosphatidylethanolamine (16%).and phosphatidylcholine (1.9%)
constitute the polar head groups of neutral phospholipids. Acidic phospholipids contain
phosphatidic acid (52.7% )and phosphatidylserine (47.3%) H. pylori does not possess
the methyl-substituted menaquinones (thermo plasma quinones ) present in
Campylobacter species. Cholesterol glycosides are a unique characteristic of the lipid
content of various Helicobacter sp.(34,52)
1.2.5 culture and colony morphology :
H. pylori is strictly microaerophilic and Co2 (5-20%)and high humidity are required for
growth.(34,53 ) H. pylori requires media containing supplements similar to those used
for Campylobacter blood .serum .starch .or charcoal. However H. pylori is inhibited by
bisulfate in the ferrous sulfite-sodium metabisulfite -sodium pyruvate (FBP)
Campylobacter ‘aerotolerance’ supplement .(34)
The good growth has been obtained by using Brucella, brain-heat infusion (BHI) and
cryptic Soya agar with 5% sheep blood and using of modified Thayer- martin agar as a
selective media for isolation of Helicobacter pylori in mixed cultures incubated at
37oC.in humid microphilics environment (5% O and 10% Co2 and 85% N2 ) for 3 - 6
days.(53)
Poor growth is observed on commercially prepared chocolate agar and there for this
media is not recommended because Helicobacter pylori is susceptible to cephalothin
and will not grow on any selective media containing cephalosporin.
7
The colonies of Helicobacter pylori are small, they grow bigger than 2mm in diameter
if incubation extended beyond 1week. gray, translucent, convex (34,53) weekly Bhemolytic. on 5%horse blood agar. motility is weak or absent when grow on agar.(34)
Gram stain reveals pale-staining curved gram-negative with characteristic gull-wing
and U shapes.
1.2.6 Pathogenesis :
H. pylori does not invade the host tissue but rather is restricted to the mucus overlaying
the gastric epithelium. During its colonization of the gastric mucosa H. pylori is
challenged by the innate and adaptive immune response, and the organism has
developed strategies of avoidance and subversion of the immune system. The virulence
factors described above support the organism’s ability to establish a persistent
infection, but mechanisms of immune evasion such as antigenic shedding of urease,
antigenic variation and a possible immune suppression by H .pylori may contribute as
well .
Urease in an important immunodominant protein that makes its way to the cell surface,
where it is not covalently attached (54), and free urease or urease adsorbed to bacteria
has been detected in the gastric mucosa of H. pylori infected patients (55,56). Shed
urease could bind to secretory IgA and thereby overcome its antibacterial activity .
After the sequencing of the H. pylori genome, sequence similarities among a group of
outer membrane proteins have become evident. It seems feasible that recombination
events could lead to mosaic organization and thus contribute to antigenic variation (57)
In vitro experiments (58,59) have shown that the proliferation of peripheral blood
mononuclear cells is decreased in H. pylori infected patients compared to healthy blood
donors. Thus some researchers have argued that H. pylori infection might lead to a
suppression of the host cellular immune response.
Both bacterial virulence factors and damage caused by the host inflammatory immune
response contribute to the pathogenesis of H. pylori gastritis .
Direct cell damage can be caused by vacA and urease, which both lead to vacuolisation
of the gastric epithelial cell in vitro .
Indirect damage to the host is caused by a vigorous immune response. H. pylori
activates the proinflammatory transcription factor NF.B in gastric epithelial and
8
monocytic cells, probably via CagA dependent and independent pathways in different
cell types (60,61). This pathway of NF.B activation, together with other pathways,
results in epithelial production of IL-8, a chemotactic factor for human neutrophils
(61,62). The chemotactic effect of IL-8, together with a chemotactic effect of H. pylori
surface proteins (55), leads to an infiltration of the gastric mucosa with neutrophils and
monocytes, causing inflammation and mucosal damage .
H. pylori adheres to and is phagocytosed by the infiltrating cells. Since it has been
shown that H. pylori is able to activate a respiratory burst in human neutrophils and
monocytes (63,64) mucosal damage may be due to the oxygen radicals produced in this
reaction .
Additionally, molecular mimicry between H. pylori LPS and host antigens might
contribute to mucosal damage through autoimmune reactions. The LPS of 85% of H.
pylori strains is composed of Lewis x and/or Lewis y antigens, human blood group
glycoantigens whose epitopes may also be present on the beta chain of the H+/K+ATPase of the human gastric parietal cell. Thus H. pylori LPS is able to stimulate
strong anti-Lewis x and anti-Lewis y responses in humans (65,66). These findings
suggest that molecular mimicry-induced autoimmune reactions could contribute to the
pathogenesis of H. pylori infection .
There are data suggesting that H. pylori affects serum levels of gastrin, a peptide
hormone that is produced by gastric G-cells and stimulates gastric acid secretion.
Studies comparing the serum gastrin levels of healthy and H. pylori-infected
individuals indicate that infection is correlated with increased serum gastrin levels.
However, the acid secretion was only elevated in patients presenting with duodenal
ulcers compared to healthy control individuals, while it was normal in asymptomatic H.
pylori-infected patients (67). This increased acid production might also contribute to
mucosal damage.
1.2.7 Diseases :
Diseases associated with H. pylori include chronic gastritis, peptic ulcer disease, gastric
cancer and MALT-lymphoma .
Today the bacterium is considered the main causative agent of chronic type B gastritis
(68)
The inflammation primarily affects the antrum and is characterized by neutrophilic and
9
lymphocytic infiltrates and distinctive epithelial degenerative changes (69).
H. pylori is an important risk factor for peptic ulcer disease, and infection has been
diagnosed in 60%-100% of gastric and 90%-100% of duodenal ulcers cases, although
in the duodenum the bacterium has only been detected in areas of gastric metaplasia
indicating its tissue specifity (70). Eradication of H. pylori from H. pylori-positive
peptic ulcer patients provides the most effective treatment resulting in ulcer healing and
low frequencies of relapse (71,72)
The chronic inflammation caused by H. pylori can result in mucosal atrophy,
metaplasia and dysplasia, thereby creating the bases for a malignant transformation
(73,74). Therefore Helicobacter infection is associated with an increased risk of gastric
cancer (75,76) .
Clinical and epidemiological studies provide data for an association of the bacterium
and MALT lymphoma. H. pylori infection has been diagnosed in over 90% of MALT
lymphoma patients (77), and antimicrobial eradication therapy can result in tumor
remission in clinical studies (78). In vitro, the growth of isolated lymphoma cells
required a T cell activation induced by H. pylori (79,80)
1.2.8 Diagnosis:
Various tests are available to assist in diagnosis of H. pylori infection .These test can
be categorized into those that are based on direct assessment of gastric biopsies
(invasive) and indirect tests (noninvasive )that detect an immunological response (i.e.
antibodies against H. pylori ) or metabolic products (i.e.urease activity) of H. pylori
(34)
The gold standard for the detection of H. pylori has been defined as culture, or
histopathological examination of gastric biopsies. Depending on the experience of the
investigator .In order to optimize the diagnosis of H. pylori .It is usually recommended
that several tests be used together .The choice of a particular test will depend on locally
available facilities, cost considerations, and clinical circumstances in which the
diagnosis of H. pylori is to be made.
1.2.8.1 Histology:
Histology is currently the gold standard for diagnosing H. pylori infection. The organ
ism can be identified on a routine hematoxylin-eosin stain but special stain
10
(e.g.Geimsa, Warthin-starry )may be even more accurate .At least two samples should
be evaluated, regardless of the stain used . Because H. pylori infection causes antral
inflammation ,some investigators suggest that absence of inflammation on biopsy is the
most dependable finding to use for exclusion of infection (negative predictive value
100%) (3,81) .
1.2.8.2 Culture :
The most specific method to diagnose infection is culture of H. pylori from gastric
biopsies, but its sensitivity depends on both the biopsy transport conditions and
expertise available in the individual laboratory (34,82). However, H. pylori is a
fastidious, slow growing organism that requires special culture conditions. So relying
on culture alone can mean missing a proportion of infected cases for technical reason,
Including overgrowth of other bacteria and low bacterial load.
Attention to transport conditions of biopsies is particularly crucial for an adequate
diagnostic yield . Biopsy specimens should be transported in a moist state, must be
cultured within 4-6h of being taken. Storage beyond this time should be at 4oC, or at
20oC if the period is more than 2days. Specimens can be kept in Stuart semisolid
transport medium for 24h if stored at 4oC (83).No transport medium is used
universally, but the various suggested transport media for biopsy samples, include
normalsalne,20%glucose,and Stuarts medium(84).An in-house Helicobacter transport
medium used successfully for several years for both biopsies and cultures comprises
brain heart infusion broth (3.5%),yeast extract (2.4%) sterile horse serum (5%)and
antibiotic supplement (Vancomycine , Trimethoprim ,cephsoludin, amphotericin ) (85).
Selective media should be used together with a non selective fresh blood-based agar..in
order to ensure optimal recovery of H. pylori, plates should be incubated in
microaerobic environment at 37oC under humid condition for at least 5-7 days before
being discarded. Colonies may be visible after 3-5days of incubation but may take
longer to appear on primary isolation.
1.2.8.3 Rapid urease test:
H. pylori produces such abundant urease that the enzyme can be detected in specimens
of infected gastric mucosa placed directly in a solution of urea with phenol red as an
indicator. In the original test, Christensen urea broth was used but unbuffered urea
solutions .If freshly prepared .and the use of gels instead of broth give quicker result
11
(34,86,87)A number of modifications have been described and kits are now widely
available from commercial sources (e.g. Campylobacter-like organism(clo)test ). The
test can have a sensitivity of at least 80% and specificity approaching 100% when the
reaction is positive within 1h. false positive may occur by contamination with ureaseproducing bacteria from the oral cavity .In clinical practice ,biopsy urease test result
should be read within 24h It has the advantage of diagnosing H. pylori status in most
cases soon after endoscopy, which is helpful for the clinician planning therapeutic
interventions. Test kits are accure of gastric biopsies (34,88).
1.2.8.4 Urease breath test :
The urea breath test identifies active H. pylori infection through the organism’s urease
production. The patient ingests urea labeled with either the nonradioactive isotope
carbon 13 (13C)(Breath Tek UBT for H. pylori, Meretek Diagnostics, Inc, Lafayette,
CO) or the radioactive isotope carbon 14 (14C) (PY test, Kimberly-Clark Corp, Draper,
UT). If H. pylori is present in the stomach, hydrolysis occurs and produces Labelle
carbon dioxide, which is detectable within a few minutes in the patient’s breath. The
labeled urea is typically given to the patient with a test meal to delay gastric emptying
and increase contact time with the mucosa. After urea ingestion, breath samples are
collected for up to 20 minutes by exhaling into a carbon dioxide–trapping agent.
Though the amount of radiation in the 14C urea breath test is less than daily
background radiation exposure,(89,90) the 13C test is preferred in children and
pregnant women.(91)
Recently, a new card test for 14C urea has been described that uses a flat breath card
that is read by a small analyzer, providing a nearpatient testing option in primary care
settings.
The urea breath test detects active H. pylori infection and is highly accurate, with a
weighted mean sensitivity and specificity from published trials of 94.7% and 95.7%,
respectively (92)
1.2.8.5 Serology :
Serologic testing detects the presence of specific IgG antibodies to H. pylori in a
patient’s serum. These antibodies are present in serum about 21 days after infection and
can remain present long after the organism is eradicated. They can be assessed
quantitatively using enzyme-linked immunosorbent assay (ELISA)and latex
12
agglutination techniques or qualitatively using office-based kits. Dozens of different
serologic tests are commercially available.( 89)
Advantages of the serologic tests are their wide availability, their rapid results, the fact
that they require no specialized equipment or techniques, and their low cost relative to
active tests. For these reasons, serologic tests were the mainstay of H. pylori diagnosis
for a number of years.(89)
The major disadvantage of serologic tests is that they cannot distinguish between active
infection and previous exposure to H. pylori because serologic testing detects only
antibodies, a positive serology result can occur in three very different patient
groups(93)
1. Those with detectable antibody and active H. pylori infection (true-positive for
antibody, infected).
2. Those with detectable antibody but not actively infected (true-positive for antibody,
not infected).
3. Those never infected and with no antibody detectable (false-positive result).
This distinction is critical because eradication therapy is of no clinical value in the
second and third groups. As more and more people are successfully treated for H.
pylori in a population, the ranks of the “true-positive for antibody, not infected” group
(group 2) will grow. Of course, the inability to distinguish between active and past
infection also renders serologic testing useless for confirmatory testing to ensure H.
pylori eradication following treatment to cure the infection.
This inability to distinguish between current and past infection contributes to the other
major shortcoming of serologic testing that it is less sensitive and specific than the
active noninvasive tests for H. pylori (92).
1.2.8.6 Stool antigen test :
The stool antigen test is an enzymatic immunoassay (ELISA) that identifies H. pylori
Antigen in stool specimens through a polyclonal anti- H. pylori antibody (Premier
Platinum HpSA, Meridian Bioscience, Inc, Cincinnati, OH). In addition, a rapid stool
antigen test (Immuno card STAT! HpSA, Meridian Bioscience, Inc, Cincinnati, OH) is
available. Using the rapid assay, a diluted stool sample from the patient is dispensed
into the sample port of the test device; after 5 minutes of incubation at room
temperature, the device indicates a positive or negative result, providing a near-patient
testing option in primary care settings(89).
13
The ELISA stool antigen test detects active H. pylori infection and is highly accurate,
with a weighted mean sensitivity and specificity from published trials of 93.1% and
92.8%,respectively (89,92) rates that are virtually the same as those for the urea breath
test.
Islam et al. (2005) evaluated the usefulness of HpSA before and after eradication
therapy on 127 adult patients. All patients underwent HpSA testing and gastroscopy
with biopsies. Those who were found to be H. pylori-positive received triple therapy
of clarithromycin, amoxicillin and omeprazole for seven days. HpSA was compared
with histology, urease testing, or a combination of the two for pretherapy testing.
After a period of six to eight weeks, post-treatment testing for confirmation of
eradication was done using the UBT and HpSA. The sensitivity and specificity were
79% and 92%, respectively, for HpSA when compared to combined urease and
histology as the reference standards. A sensitivity of 67% and specificity of 100%
were reported for HpSA when compared to UBT used as the reference standard for
post-treatment testing. The authors concluded that “in this prospective study, HpSA
was found to be a reasonably useful diagnostic test for H. pylori infection .(94,95)
1.2.8.7 Near patient test :
Rapid office-based tests using whole blood have been developed as they are quick and
easy to perform. but sensitivity and specificity vary widely .Their inaccuracy therefore
precludes there use as diagnostic test in genral practice setting(96).
1.2.8.8 Salivary and urine antibody :
The use of commercial assays to detect anti-H. pylori IgG antibodies in saliva or urine
are attractive alternatives as they are completely noninvasive and easy to perform. The
accuracy is unacceptable (sensitivity 81% and specifity 73% ) (97).However .data on
urine antibodies assay indicate an accuracy comparable to that of the serum ELISA in
Japanese (98) and European population (96).
1.2.8.9 Molecular test :
There are no commercially available assays for the detection of H. pylori in gastric
biopsies even though several validated genus and species-specific probe hybridization
and PCR assays have been described and have recently been reviewed (99) .The
method with the potential for greatest sensitivity is detection of H. pylori-specific
DNA by PCR .Gastric biopsy material and gastric juice aspirates have been used for
14
PCR diagnosis targeting various DNA sequences including regions of 16S rRNA
gene (100). urease subunit genes (101).The vacA gen (102).and a portion of a gene
encoding a spescies-specific 26 kDa protein (103).Results with these PCR tests have
yielded ranges of sensitivities and specificities depending on the material analyzed
and the assays (99).
Molecular techniques may be become more relevant for the diagnosis of H. pylori.
1.2.8.10 String test :
The swallowed string test (Enterotest ) is minimal invasive approach as an alternative
to endoscopy for direct sampling of gastric fluid, although there is no information on
its use in clinical practice . samling of gastric juice provides a good measure of the
infecting strain population shed from the gastric mucosa (104) so analysis of the string
sample by culture or PCR could be informative when its accuracy has been
established.(34).
1.2.9 Immunity:
Patients infected with H pylori develop an IgM antibody response to the infection .
Subsequently, IgG and IgA are produced, and these persist, both systemically and at
the mucosa in high titer in chronic infected patents
1.2.10 Transmission:
Its transmission has not been completely understood. Direct person to person
transmission is considered to be the most likely mode and it is believed that the
transmission either follows the fecal-oral or the oral-oral route. Fecal-oral (1,38,131)
transmission gained importance by the detection of H. pylori in the faeces of infected
patients (132), while studies comparing the distribution patterns of H. pylori with
Hepatitis A virus, an organism transmitted by the fecal-oral route, suggest that this
transmission mode may be of limited importance (133). There is evidence that
acquisition of infection takes place predominantly in early childhood (134), and the
host usually remains infected for a lifetime.
Transmission of the bacterium among adults seems less frequent although it seems
favoured when there is close personal contact, as there are higher prevalences in
institutions (135).
15
1.2.11 Epidemiology:
childhood (136,137), close person to person contact (138,139), and low socioeconomic
status (140). Thus the prevalence of H. pylori infection ranges from approximately 20
to 50% in industrialized countries to over 90% in developing nations (141,142). As
determined in a study of the Robert Koch Institute in 2000, the prevalence of H. pylori
infection for the total population of Germany is 40% (143) It is estimated that 50% of
the world’s population is infected with H. pylori. Infection rates ،like for many other
infectious diseases, correlate with poor living conditions H. pylori is present on the
gastric mucosa of less than 20% of persons under age 30 but increases in prevalence to
40-60% of persons age 60 including persons who are asymptomatic. In developing
countries the prevalence of infection may be 80 %or higher in adult.
\
1.2.12 Treatment:
Eradication of H. pylori is probably one of the most frequently prescribed antibiotic
based complex therapies due to the widespread rate of colonization of the bacteria in
humans. management of H. pylori infection in different gastrointestinal diseases is
guided by international and national guidelines and recommendations (e.g. Maastricht
Consensus reports for Europe).(105) .
At present, clarithromycin (Cla), amoxicillin (Am) and proton pump inhibitor (PPi)
based triple therapy regimen constitutes the most generally accepted first line empirical
treatment for H. pylori infected patients world-wide. However, the growing number of
reported therapy failures has long prompted clinicians to seek more effective solutions.
(106.107). These efforts, based mostly on empirical observations with different
antibiotic combinations, have not led to widely accepted, conclusive therapy protocols,
so far.
When selecting a therapy to eradicate H. pylori, duration of treatment and adverse
effects should be considered (108)
Until recently, the recommended duration of therapy for H. pylori eradication was 10
to 14 days. The most widely recommended regimens are summarized in.37Studies
evaluating one-, five-, and seven-day regimens to eradicate H. pylori are summarized
in(108,109,110).
Although not proven, potential benefits of shorter regimens include better compliance,
fewer adverse drug effects, and reduced cost to the patient.(108)
16
1.2.12.1 Firest-line treatment :
The best validated first-line treatments for H. pylori include clarithromycin-based triple
therapies consisting of a proton pump inhibitor (PPI), clarithromycin, and either
amoxicillin or metronidazole and bismuth-based quadruple therapies consisting of a
histamine receptor antagonist or PPI combined with bismuth, tetracycline, and
metronidazole. Where available, ranitidine bismuth citrate (RBC) can be substituted for
a PPI in clariithromycin triple therapy .These regimens currently yield eradication rates
ranging from 70%–85%. Triple therapy employing a PPI with clarithromycin and
amoxicillin is the most widely endorsed first-line regimen for H. pylori
eradication(111,112,113)
Any of the currently available PPIs may be used with equivalent treatment
(114,115)With the exception of esomeprazole which can be prescribed once daily, it is
important that the standard dose of a PPI be prescribed twice daily to maximize
treatment efficacy.(116) RBC is sometimes used in place of a PPI in countries outside
the United States (where it is not commercially available) with at least equal and
perhaps greater efficacy.(117)
Metronidazole can be used as an alternative to amoxicillin, particularly Bismuth-based
quadruple therapy is another option in penicillin allergic patients which yields similar
eradication rates to clarithromycin triple therapies(.118,119,120) .
Recently, simplified twice-daily dosing regimens for bismuth quadruple therapy have
been successfully used in clinical trials.(121) It is worth noting that the dosing of
metronidazole used in the various bismuth quadruple therapies has not been entirely
consistent across studies.
As higher doses of metronidazole (500 mg) may provide better cure rates than lower
doses (250 mg),caution must be exercised when interpreting the data from comparative
studies and pooled analyses involving quadruple therapies.
Administration for use in the United States. A recent meta-analysis of 7 studies
involving more than 900 patients found that a 14-day course of clarithromycin triple
therapy provided better eradication rates than a 7-day course of therapy. There was also
a trend toward improved efficacy with10 days of therapy compared to 7 days of
therapy which did not reach statistical significance.(122) Due to falling eradication
rates with clarithromycin-based triple therapy, it is essential to take every opportunity
to optimize treatment success. clarithromycin triple therapy, particularly in regions
such as the United States where eradication rates have been 80% or less with shorter
durations of therapy. Although some promising data exists on the effectiveness of
17
ultra short course of therapy,(111,123,124) treatment duration of less than 7 days
cannot currently be recommended
1.2.12.2 Second-line therapies :
Even with the current most effective treatment regimens, About 10-20% of the patients
will fail to eradicate the infection and will remain H. pylori positive after 4 weeks or
longer .Failures are due mainly to bacterial resistance or poor compliance. Widespread
resistance to antibiotics used in eradication therapy. In particular metronidazole and
clarithromycin, is a source of growing concern because of its impact on eradication
.The clinical relevance of antibiotic resistance is increasingly accepted as an issue and
its importance in therapy has been high lighted in several recent studies (125,126) .
An optimal strategy for retreatment after initial failure of eradication has not yet been
established, but the recommended second line therapy according to the Maastricht II2000 report is use of quadruple therapy a PPI with bismuth ,metronidazole ,and
tetracycline (an antibiotic un likely to have been used previously in front-line therapy ).
It is advisable that the antibiotic susceptibility of the organism is determined before
retreating because of high resistance rates in some populations to metronidazole .
The use of ranitidine-bismuth-citrate-based triple therapy is another possibility for
retreatment (127).
1.2.12.2 Resistance:
Although first-line therapy will successfully eradicate the bacteria in most infected
patients, antibiotic resistance of H. pylori is a growing concern.(128,129 ).Resistant H.
pylori has been documented in cases of failed eradication therapy based on biopsy and
culture results and is of great concern in patients at high risk for complications of H.
pylori infection.
In one small trial, 70 percent of patients failing one or more regimens responded well
to triple-drug therapy that included pantoprazole (Protonix), amoxicillin, and
levofloxacin (Levaquin) for 10 days (130) A meta analysis of current literature on
treatment of resistant H. pylori showed some benefit in using quadruple drug therapy,
including the addition of clarithromycin to ranitidine (Zantac),bismuth, and amoxicillin
(1g twice daily)therapy, as well as a combination of proton-pump inhibitors (standard
dosage for 10 days),bismuth, metronidazole (Flagyl), and tetracycline. (129)
18
Regimens that include rifabutin (Mycobutin), 300 mg per day, also have been
successful in 38 percent of resistant cases.(128).
1.3.1 Medicinal plants:
The medicinal plants were known since old ages and were handled by many people as
traditional medicine to treat many diseases.
Statistical purposes to use these traditional medicines were found very high in
developing countries and Sudan is one of them.
To use traditional medicine which was very primitive not scientifically studied for the
right quality and quantity and the side effect on users, there are many scientific
institution and researchers as individual their attention to study many plants that are
used in traditional medicine, to extract the useful ingredient without the harmful
material. Commiphora myrrh is one of the medicine plants that are commonly used in
Sudan in traditional medicine for the treatment of many diseases. This plant is found in
great quantity in North-East Africa, collected in Southern Arabia and Iran.
[
1.3.2. The activity of medicine plants:
Effective efforts have been made over last fifty-year to find new antimicrobial agents
the major part of the reported investigations was concerned with lower plants with
special attention being paid to different species of Streptomycets and some fungi .
However, a number of reports indicated new source of anti-microbial agents from
higher plants (144)
Beloy,et al(1976) studied 106 species belonging to44 families for antimicrobial activity
9 plants showed high antimicrobial activity
Thirty-three plants of Pakistan origin screened by Ikram and Inamul-Hag(1980) for
antimicrobial activity (144).
Wondergam and grand (1984) examined 33 species (used against infectious diseases
by the diolaof southern Senegal) for antimicrobial activity. Of these 28were active
against such organisms as Staphylococcus aureus, Bacillus subtilis and Asperigillus
niger (144)..
Forty-five species of Somalian medicinal plants screened by Elmi et al (1986) for
antimicrobial activity against (Staphylococcus aureus, Bacillus subtilis) They found
19
that, thirty species showed activity against Gram-positive bacteria . (144).
Elagimi et al (1998) screened 114 extracts of Sudanese plants used in folkloric
medicine for their anti-bacterial activity against (E. coli, Staphylococcus aureus ,
Bacillus subtilis , P. aeruginosa) Of these,83,(73%) extracts showed significant antibacterial activity. (144).
1.3.3. Commiphora myrrh :
As mentioned by Guenther(145), myrrh (also called heerabol-myrrh or bitter myrrh)
is gum-resin obtained from several species of Commiphora (fam. Burseraceae),
notably C. Abyssinic(Berg) Engler, oofC. schimperi (Berg) Engler, and C. myrrh
(Nees) Engler var molmol Engler. Genus Commiphora comprises more than 200
species, all native to Africa, Arabia, Madagascar and India. In order to collect gum,
the natives make incisions into the bark, causing the exudation of a yellowish oleo
resin. Exposed to the air, this dries, hardens and turns reddish-brown..(146)
Myrrh is partly soluble in ethanol (30 % alcohol soluble material) and is also partly
soluble in water and in ether. Since antiquity myrrh has served as a constituent of
incense. Oil of myrrh is a valuable ingredient in perfumes (balsamic, heavy odour).
The chemistry of myrrh..(146).
Commiphora myrrh (also known as Commiphora molmol and Balsamodendron myrrh)
of the Burseraceae family and is also known as bola, Myrrh is a large shrub or small
tree that grows in the Middle East and Ethiopia and Somalia. A pale yellow oil drips
from the cuts in its dull gray bark and hardens to form teardrop-shaped nuggets of
myrrh, which are powdered for use as a healing herb. (147)
Myrrh is referred to in the Bible. It was used by Egyptians in embalming mixtures. It
was used as an aromatic for perfumes, funerals, and insect repellents. It is used today as
an aid to repel tooth decay and gum disease. Ancient Greek and Roman physicians
used the herb to treat wounds and prescribed it internally as a digestive aid and
menstruation promoter (147,148).
Contemporary herbalists recommend adding powdered myrrh to well-washed wounds
as an antiseptic and consider a gargle made from the herb effective against sore throat,
colds, sore teeth and gums, coughs, asthma, and chest congestion.
Anti-microbial, astringent, carminative, anti-catarrhal,expectorant, analgesic,antiseptic,
antispasmodic, emmenagogue, expectorant, stimulant, rejuvenative.
20
Myrrh is an effective anti-microbial agent that has been shown to work in two
complementary ways. Primarily it stimulates the production of white blood corpuscles
(with their anti-pathogenic actions) and secondarily it has a direct anti-microbial effect.
Myrrh may be used in a wide range of conditions where an anti-microbial agent is
needed. It finds specific use in the treatment of infections in the mouth such as mouth
ulcers, gingivitis, phyorrhoea, as well as the catarrhal problems of pharyngitis and
sinusitis. It may also help with laryngitis and respiratory complaints.
Systemically it is of value in the treatment of boils and similar conditions as well as
glandular fever and brucellosis. It is often used as part of an approach to the treatment
of the common cold. Externally it will be healing and antiseptic for wounds and
abrasions.
Myrrh is a common ingredient in European toothpaste to fight the bacteria that cause
tooth decay.
Used in traditional Chinese medicinal herbs as antineoplastic agents and antitumor
Myrrh may help prevent heart disease. Preliminary Indian studies suggest that it
reduces cholesterol. The herb may also help prevent the internal blood clots that trigger
heart attack.
Myrrh appears in British Pharmacopoeia and other modern pharmacopoeias (149)
Tincture of myrrh, prepared by extraction with 90% alcohol,(150)is used in
mouthwashes and gargles for its astringent effect on mucous membranes ,particularly
for mouth ulcers, gingivitis and pharyngitis. It has also been applied to the skin for
wounds and abrasion (151) although there are reports of contact dermatitis developing
as a result of topical application of myrrh –containing plasters (152).Other myrrh –
containing products include toothpastes ,hairsprays, perfumes, aromatherapy oils and
toiletries (153). Myrrh is also permitted to be used in foodstuffs in small
quantities(154).Teething-gels containing myrrh have been used to relieve teething pain
in infants(155).
One of the newest developments in the uses of myrrh has also been the most
controversial and relates to its role in treating Schistosomiasis (148).
21
1.3.3.1 Active Compounds:
Volatile oil, containing heerabolene, cadinene , elemol , eugenol cuminaldehyde,
numerous furanosesquiterpenes including furanodiene, furanodienone, curzerenone,
lindestrene, 2-methoxy furanodiene and other derivatives.
Resins including a-, b- and g-commiphoric acids, commiphorinic acid, heeraboresene,
a-and b-heerabomyrrhols and comm
Gums, composed of arabinose, galactose, xylose and 4-O- methylglucuronic acid
22
Objective of the study
General:
To study the effects of Commiphora myrrh in clinical isolate of Helicobacter pylori
from peptic ulcer patient .
Specific:
1.
To determine The prevalence of Helicobacter pylori in G I.T. patients.
2.
To Identify the isolates using standard techniques.
3.
To Conduct sensitivity test to the Commiphora myrrh in comparison
used antimicrobial agents with .
.
23
Materials and methods
2-1 Study approach:
Quantitative study aimed to identify causative agents of peptic ulcer disease and their
susceptibility testing against Commiphora myrrh and commonly used antimicrobial
agents
2.2 Study design :
The study was designed as a descriptive study
2.3 Study area :
The study area was facility based conducted in Khartoum state at Ibn Sina
endoscopy department
2-4 Study variables:
Frequency of peptic ulcer disease, antimicrobial sensitivity.
2.5 Study population:
A total number of hundred patients suffering from gastritis, duodenitis, and peptic
ulcer in Khartoum state at Ibn Sina hospital attending to endoscopy department
Were included in this study.
2.6 Selection criteria :
The selection criteria depend on the hospital attendance (in and out patients).
only patients responding to the questionnaire
2.7 Ethical clearance:
- Approval was obtained from University, Faculty and Department.
- The consent was obtained ibn sina endoscopy unit hospital and from patients.
2.8 Material:
3.8.1 Culture media:
Brain Heart Infusion broth
Brain Heart agar
Mueller Hinton Agar
Oxoid – England
Oxoid – England
Oxoid - England
24
Exp 2/2008
Exp 2008
Exp 20011
2.8.2 Supplement :
Skirrows supplement:
Vancomycin
Polymyxin bsulphate
Trimethoprim lactate
Amphotericin B
Exp 2008
(10 mg/l)
(250-300mg/l)
(5mg /l)
(2mg/l)
2.8.3 Antimicrobial disk
Amoxicillin
Tetracycline
Metronidazole
Clarithromycin
(30 ug)
(30 ug)
(32 ug)
(15 ug)
3/2008
3/2008
3/2008
3/2008
2.8.4 Chemicals & biological materials :
Gram stain (Crystal violet,Gram Iodine ,Safranin,Ethyl Alcohol.)
Hydrogen peroxide
Glycerol
Urease reagent (Urea ,Phenol red)
Oxidase discs(tetramethyl-p&phenylene-diamine dihydrochloride )
Microaerophillic systems gas generation kits
Horse serum
Sheep blood
2.8.5 Instruments &glass ware :
Autoclave
Anaerobic jar
Hot air oven
Microscope
Incubator
Sterile plates
Sterile pipettes
25
Sterile containers
Sterile tubes
Tube racks
Standard loop
Slides
2.9 Methods:
2.9.1 Collection of the samples :
The samples were taken from 100 PUD patients by endoscopy at Ibn Sina Hospital in
the period September 2006 to November 2007.
Two biopsy specimens were taken from the antrum of the PUD patients one for rapid
urease test on urea media and the other for culture on brain heart infusion broth media.
2 .9. 2 Rapid urease test :
In sterile container 1 ml of urea media with few drops of phenol red as indicator was
Placed .One of the two biopsy was placed in the mixture .the result observed by the
change of the colour from yellow to pink due to break down of urea by urease enzyme
which is produced by the organism to give ammonia and carbon dioxide which changes
the media PH to alkaline that means the organism is H. pylori .
Urease test vary according to the volumes of broth or agar, concentration of urea and
phenol, and incubation temperature.
2.9.3 Culture:
Isolation of H. pylori by culture is available diagnostic technique even a small number
of organisms in the biopsy specimen is sufficient to provide a positive culture. To
obtain optimal result it necessary to take great care in the preparation and
transportation of the specimen (3). A culture can be performed on solid or liquid media
.In clinical practice, a culture is required mainly if antibiotic sensitivity is to be tested .
To isolate H. pylori the other biopsy specimen was placed in brain heart
infusion broth as transport media ,then it was homogenized and inoculated in brain
heart agar supplemented with Skirros’s supplement (Vancomycin, Polymyxin B
sulphate, Trimethoprim lactate )and sheep or horse blood and incubated for 5-7 days
under microaerophllic condition (156 ) campy pack systems.(34)
26
The organisms were identified by colonial morphology, gram stain and biochemical
tests (urease, catalase and oxidase ).
2. 9.4 Identification of the isolates:
2 .9.4.1 Colonial morphology
The significant growth of colonies were examined morphologically for size,
Consistency, convex and ability to haemolysis .
2.9.4.2 Gram staining :
The gram staining reaction used to help identify pathogens in specimens and cultures
by their gram reaction (gram positive or gram negative ) and morphology .(157).
Gram stain used in routine identification , the bacteria film fixed and stained with
triphenyl methane dye basic dye such as crystal violet treatment with iodine solution
and subsequently treated with alcohol, those bacteria which retain the dye are gram
positive and those varieties of bacteria which loose the dye are gram-negative
(3,34,157).Decolorized cells were stained by counter stain such as safranin red . The
organisms were smeared in clean slid. The smears were allowed to dry in air and then
fixed by gentle flaming. The slides were placed on the staining rack and flooded with
crystal violet stain for one minute, Washed rapidly with clean tap water, covered with
lugol`s iodine for one minute , Washed rapidly with clean tap water .All the smears
decolorized rapidly with alcohol (few second ) and wash immediately with clean tap
water ,The smears covered with counter stain neutral red for one minute. The stain was
washed with clean tap water.
The slides were placed in the slides rack for the smear to air dry ,Then the smears were
examined microscopically using oil immersion objective and the results reported
The organisms were gram negative bacilli .
2.9.4.3 Urease test
Testing of urease enzyme activity is important to differentiating the urease
producers bacteria.
The test organism is cultured in medium which contains urea and the
indicator phenol red . when the strain is urease producing the enzyme will break down
the urea by hydrolysis to give ammonia and carbon dioxide.
27
With the release of ammonia medium change to alkaline as shown by change in colour
of indicator to pink colour(157)
In this work some colonies from each isolate were emulsified in 1 ml of urea reagent
with phenol red in sterile tube, the organism which were urease producer will
hydrolyze the urea to give ammonia and change the PH to alkaline to produce pink
colour.
2.9.4.4 Catalase test :
This demonstrates the presence of catalase enzyme that catalyses oxygen from hydrogen
peroxide (157).and used to differentiate those bacteria that produce the catalase
enzyme(157).
A colony was picked by platinum loop or wooden stick and placed in 3% hydrogen
peroxide solution in sterile tube
Catalase enzyme acts as a catalyst in the break down of hydrogen peroxide to oxygen
and water with immediate effervescence occurred which indicated positive result.
2.9.4.5 Oxidase test:
This test depends on presence in bacteria of certain oxidases that will catalyze the
transport of electrons between electron donors in the bacteria and a redox dyetetramethyl-p-phenylene-diamine. The dye is reduced to deep purple color(164).
The isolates were smeared in oxidase reagent strip the phenylene-diamine in the
reagent oxidized to deep purple color(157).
2.10 Sensitivity test :
Laboratory antimicrobial sensitivity testing can be performed using
The dilution technique
The disc diffusion technique.
In this work the two methods were used .
2.10.1 Inoculums’ preparation:
H. pylori strain from gastric biopsy specimens were isolated on brain heart agar and
stored at - 80 0C in glycerol media (Brain heart broth with 25% glycerol).
The isolate were incubated in brain heart agar supplemented with 10% horse blood
(158) for 48h at 37oC under microaerophilic condition ,produced as described above.
Given the importance of inoculum homogeneity (158,159), cellular viability was
28
controlled microscopically by morphological observation with gram stain, cultures
were always used after 48h of incubation when they generally didn't present coccoid
forms(160) Suspensions were prepared in sterile normal saline to an opacity of
McFarland standard (ca109cFU/ml).(158.161).
2.10.2. Disc diffusion method :
Disc diffusion techniques are used by most laboratories to test routinely for
antimicrobial sensitivity. A disk of blotting paper is impregnated with a known volume
and appropriate concentration of antimicrobial. not more than six discs should be tested
on a single plate to avoid fusion of inhibition zones to each other.
Modified Kirby-Bauer sensitivity testing technique was used Mueller Hinton
sensitivity testing agar prepared and sterilized and cooled then treated with horse serum
and poured in sterile Petri dishes to depth of 4mm. The prepared media was solidify
and dried (162).
The prepared inoculums were inoculated in the media with sterile loop.
The antimicrobial discs (Amoxicillin, Claritheromycin, Metronidazole, Tetracycline).
Were picked by sterile forceps and places on the surface of the inoculated media and
pressed lightly by the forceps to make contact with media to allow the antimicrobial to
diffuse from the disc to the media .
The plates ware incubated at 370C for 2-3 dayes under microaerophilic condition.
(Campy-pak system).The culture was examined for zones of inhibition of bacterial
growth around the respective disks.
2.10.4 Agar dilution method:
The principle of the agar plate dilution method is the inhibition of growth on the
surface of the agar by the antimicrobial agent (plant extract) incorporated into the
medium.
The organisms tested were prepared in sterile normal saline to an opacity of McFarland
standard (ca109cFU/ml).(158,161).
A loop full of the diluted culture is spotted with a standard loop that delivers 0.001ml
on the surface of the media .
Sterile screw cap tubes were numbered .All the following steps were cared out using
aseptic technique.
1ml of Commiphora myrrh extract was added to 10 ml of methanol as the first tube and
10ml of sterile distilled water was added to all other tube.
29
10 ml transferred from the first tube to the second tube and mixed well with separate
pipette and transferred 10 ml to the third tube.
The dilution was continued in this manner to tube number 5and the pipettes changed
between tube to tube to prevent carry over of extract on the external surface of the
pipette .10 ml removed from tube 6 and discarded .
Tube 6 which served as a control, no Commiphora added to it.
Muller-Hinton media prepared in dabble concentration for amount of the media to 20
ml of D.W only 10 ml were used and autoclaved and cooled and supplemented with
horse serum and the diluted extract added to it , mixed and transferred to the plate.
After one drop of the suspension transferred to the plate .Incubated at 370C
for two days and the result observed by growth or no growth
The highest dilution without growth is the minimum inhibitory concentration
2.11 Data analysis:
The data collected was analyzed using statistical package for Social Science for
Personal Computer (SPSS/PC). Difference was considered significant at P value
<0,05 and 95% Confidence Interval
.
30
Results
Hundred Specimens of antrum biopsy were taken by physician from Sudanese
patients with Peptic ulcer disease who attended during the period October 2006November 2007
3.1 Demographic features of study population:3.1.1 Gender And Age :
Demographic features of the patients included in the study 52 female and 48.
(fig 1) and age ranged between 19 – 70 years which as classified in the study to
be > 40 =64% and <40 =36 % .(Table 1) .
3.1.2 Residence distribution:Most of the patients were from Khartoum state 62%, 14% from the North, 5% from the
East Sudan and from the West Sudan 4% and 10 from Southern Sudan and from
Central Sudan 5% .( fig 2)
3.1.3 Occupation distribution:
The study population distributed according to there jobs to 32 manual workers, 31
house wives and 15 students , 6 farmers and 16 other jobs (Doctors ,Employers
,Engineers and Teachers). (fig 3).
3.2 Endoscopy results:
The study population included 15 Gastritis, 4 Duodenitis, 32 both Gastritis &
Duodenitis, 14 Duodenum ulcer ,5 Gastric ulcer and 2 with Esophagus variesis and 5
with both Gastric ulcer & Duodenum ulcer ,2 were Gastric hernia 8 with both Gastritis
& Duodenum ulcer and 3 with Gastritis & Esophagitis & duodenum ulcer 7 were with
Gastritis and esophagitis .(fig 4)
[
3.3 Rapid urease test results:
In the hundred specimen 86% were showed positive rapid urease test and 14%
31
negative for urease test. (Table 2).
3. 4 Culture results:
Sixty showed positive growth and fourty were negative growth .(Table 3)
60 of urease positive showed positive growth and 26 no growth, none of negative
urease showed growth (table 4).
The growth rate in women was higher than in men 53.4%: 46.6 % respectively (table 5)
In the study population there were high positive growth in house wife 23, manual
workers 19 , 7 students and 5 farmers and 6 others ( table 6) .
Positive culture growth were high in Khartoum state (Table 7).
Endoscopy findings showed high growth rate in Gastritis & duodenitis 21 and
duodenum ulcer 9 and 7 and Gastritis duodenal ulcer and gastritis 5and 4 Gastric
ulcer & duodenal ulcer And 3 duodenitis and 3 gastric ulcer and the same number
Gastritis & esophagitis .& duodenal ulcer and 2 Gastritis & gastric ulcer and 2
Gastritis & esophagitis . and one esophagus varaesis and there were no growth in
esophagitis . nor in gastric hernia Fig (5).
3.5 Sensitivity test results:
3.5.1 Antibiotic disk results:
The susceptibility tests for sixty H .pylori isolates were determined by the Kirby Bauer
disk diffusion technique against(Amoxicillin, Metronidazole, Tetracycline
,Clarithromycin ).
The sensitivity assessment showed high resistance rate in Clarithromycin and
Metronidazale and high sensitivity to Amoxicillin and then to Tetracycline (fig 6, 7, 8,
9).
3.5.2 Plant antimicrobial results:
The plant antimicrobial activity was determined by agar dilution method to plant
extract against H. pylori isolates and which showed high sensitivity 100% in dilution
one (1/20) and 90% in dilution two (1/40) and in the third dilution (1/80) the
sensitivity 50% (30 out of 60) and the sensitivity decrease in dilution four to (1/160)
20% (fig10,11,12,13).
32
Fig (1)
Gender distribution and percentage in study population.
33
Table (1)
Age distribution and percentage in study population.
Age
>40
Number of patient
Percentage
64
64.0%
<40
36
36.0%
Total
100
100.0%
34
Fig (2)
Distribution of the study population according to their residence
35
Fig (3)
Distribution of the study population according to their Occupation .
36
10
0
Frequency
Endoscopy
INDOSCOP
40
30
20
it
eg
ph
se
O
&
ric
tis
st
i
tri
ga
ag
as
&
G
s
ph
e
iti
tr
os
as
al
&
G
s
en
iti
tr
od
i
as
du
en
G
od
tis
tri
du den
&
as
o
G
du
tis
is
r&
tri
ce aes
as
ul
r
G
c
va
tri
s
u
as
G
ag
ph
ia
so
rn
oe
ha
c
iri
r
st
ce
ul
r
ga
c
ce
iri
ul
st
ga num
e
od tis
du
gi
ha
sp s
oe
iti
en
od
du s
i
rit
st
ga
Endoscopy
INDOSCOP
Fig (4)
Distribution of study population according to Endoscopy results
37
Table (2)
Results of Rapid urease test in hundred GIT biopsy
Number of biopsy
Rapid urease test
positive
negative
Total
Percentage
86 %
14 %
100 %
86
14
100
38
Table (3)
Percentage of the growth in one hundred GIT biopsy
Culture
Number of biopsy
Percentage
Growth
60
60 %
No growth
40
40 %
100
100 %
Total
39
Table (4)
Relation ship between the growth and urease results.
Culture
growth
no growth
Total
Rapid urease test
positive
negative
60
0
Total
26
14
40 %
86
14
100 %
40
60 %
Table (5)
Relation between Gender In the study population and growth.
Gender
Male
Female
Total
Growth
56 %
63 %
60 %
Culture
No growth
44 %
37 %
40 %
41
Total
48
52
100
Table (6)
Relation ship between Occupation in the study population and
growth of H. pylori .
Occupation
House-wife
Manual workers
Students
Farmers
Others
Total
Culture
Growth
74%
59%
47%
83%
39%
60%
42
No growth
26%
41%
53%
17%
62%
40%
Total
31
3
15
6
16
100
Table (7)
Relation ship between residence of the study population and growth.
Residence
Khartoum
North Sudan
East Sudan
West Sudan
South Sudan
Central Sudan
Total
Culture
no
growth
growth
60 %
40 %
64 %
36 %
40 %
60 %
100 %
0 %
60 %
40 %
40 %
60 %
60 %
40 %
43
Total
62
14
5
4
10
5
100
grow th
no grow th
0
Count
30
20
CULTURE
10
it
eg
ph
se
O ic
&
r
t
ti s as gi
g
tri
a
as s & ph
G
i
e
t
os al
tri
n
&
as
G iti s ode i
n
u
tr
as s d ode
G
i
u
t
en
tri & d o d
as
u
G ti s &d i s
tri cer
es
l
as
ra
G ic u va
s
tr
as gu
G ha ia
p
n
s o har
oe
c
r
iri
ce r
st
l
ga ic u l ce
u
ir
st m
ga enu
od i tis
du ag
h
sp is
oe nit
e
od
du tis
ri
st
ga
Endoscopy
INDOSCOP
Fig (5)
Relation ship between endoscopy finding of the study population and growth.
44
Fig (6)
Sensitivity and resistance of H. pylori to Clarithromycin.
45
Fig (7)
Sensitivity and resistance of H. pylori to Metronidazole .
46
Fig (8)
Sensitivity and resistance of H. pylori to Amoxicillin.
47
Fig (9)
Sensitivity and resistance of H. pylori to Tetracycline.
48
Fig (10)
Sensitivity and resistance of H. pylori to Commiphora myrrh extract in dilution one
(1/20)
49
Fig (11)
Sensitivity and resistance of H. pylori to Commiphora myrrh extract in dilution tow
(1/40)
50
Fig (12)
Sensitivity and resistance of H. pylori to Commiphora myrrh extract in dilution
three (1/80).
51
Fig (13 )
Sensitivity and resistance of H. pylori to Commiphora myrrh extract in dilution
four(1/160) .
52
Discussion
In the present study the prevalence of H .pylori infection observed was higher in
women than men, this confirmed what found in the studies that were done in Khartoum
teaching hospital and Academy Charity teaching hospital, Soba hospital and Ibin Sina
hospital (3,163,164) and similar to that reported in the study done in Ninth people`s
Hospital of Shanghai (165).
In this study the prevalence of H. pylori infection was high among elderly as it was
observed in the study done by Gunaid et al (166) which found that there was
significant difference in prevalence of H .pylori infection between age group 41-75
year (90%) and age group 16 -36 years (75%) (166,167) and similar to study done by
Duggan and Duggan (2006)(13) found that H . pylori colonization rates remained high
in the elderly and also in agreement with the study done in King Faisal University
(168).
In this study, although most of study population came from Khartoum state but
originally were from the rural areas of Khartoum. This agree with the study that found
urbanization and the crowded living conditions of expanding cities might result in
decline in hygiene for large sections of the population . This phenomenon may be
associated with a concomitant rise in exposure and prevalence of H . pylori infection
(166,168 ). And also in agreement with the study which it found that 60% of the
patients were originally from rural areas (3).This may explain that H .pylori infection is
common in poor areas and another study that showed the infection in rural population
had been higher than that of an urban population (169) .
In this study, most of population are manual workers (32%) , 31% were house wives
and 15 % students , 6% farmers and all other jobs (Doctors ,Employers , Engineers and
Teachers) were16%..
In this study the sensitivity of urease test was high 86% and the specificity compared
with the culture as gold standard method was 71.4% . The high sensitivity is similar to
that in study done in Sana`a (166) and also agree with the study done in Ibn Sina
Hospital (170) and similar to the study that was done by Thillanyagam et al who was
found that the sensitivity was 83% (171) and slightly high from that in study done in
South Africa 71% (172). This sensitivity did not confirm the higher sensitivity reported
by Marshall etal 1987 which was found to be 99% (173) .
53
In This study there were 32 cases of both Gastritis & Duodenitis and 15 Gastritis,
4Duodenitis, and, 14 were Duodenum ulcer and 5 with Gastric ulcer and 2 with
Esophagus variesis and 5 with both Gastric ulcer & Duodenum ulcer and 2 were
Gastric hernia 8 with both Gastritis & Duodenum ulcer and 3 with Gastritis &
Esophagitis & duodenum ulcer 7 were with Gastritis and esophagitis This showed that
the endoscopic appearance of ulcer and inflammation is acute and active in most of the
patients which agreed with the studies done in Soba Hospital and Khartoum Teaching
Hospital and Academe hospital (3, 163) .
In this study susceptibility of H . pylori to antimicrobials among Sudanese patients
with peptic ulcer disease was determined by agar dilution method for the plant
Commiphora myrrh and disk diffusion to antibiotic disks ( Metronidazole ,Amoxicillin,
Tetracycline, Clarithromycin).
Metronidazole:
The resistance of H. pylori to Metronidazole varies from population to population
(168,174,175,176) It is highest in the developing and lowest in developed countries
(174,175,177) .In this study, the prevalence of Metronidazole resistant H .pylori
isolates was 61.7 %; it is similar to the study done In Sudan at Ibn Sina Hospital which
found the resistance rate was 61% (170). This high resistance rate is similar to finding
in the study done in urban Asia population 62% (178) also the results of the present
study agreed with the results of study done in Japan which observed that a higher
prevalence of Metronidazole -resistant strains is seen in the western or southern regions
although it was very rare in northern region of Japan and in Japan generally; this was
explained differ by the close proximity of these regions to other Asian countries,
which have a high prevalence of Metronidazole -resistant H. pylori. South and West
Japan are much closer to these countries than North Japan (179).
It also agreed with the results of the study done in King Fahd Hospital of university
Khobar which found high prevalence of Metronidazole -resistant H. pylori(180) and it
was slight high than resistance rate reported from Bahrain ,Gulf region which found
those who were infected by resistant strains to Metronidazole were 57% (181.)
Resistance of H .pylori to Metronedazole is related to the previous exposre to the drug
(174,175,182) It has been suggested that the high prevalence rate of Metronidazole resistant H. pylori in women may be due to it use in the treatment of vaginal infections
such as trichomoniasis and Gardnerella and as prophylaxis in uterine surgery (182)
.Previous use for the treatment of amoebiasis and other diarrheal diseases and possible
increased use for protozoal and anaerobic infection .
54
Clarithromicin
The likelihood of antibiotic resistance varies by geographical Region.. Additionally,
prior use of clarithromycin or metronidazole for any infection, has been shown to
increase the likelihood of H pylori resistance to these agents(183). Clarithromycin
resistance varies from less than 5% in southern parts of Europe to greater than
20% in northern Europe(195) to as high as 80% in some developing countries. (184)
which was agreed with the finding of study that showed high prevalence of
Clarithromycin- resistant H .pylori ,also agreed with the study done in Japan(179) and
that one done in Singapore (185) .
Amoxicillin
Antibiotics. Amoxicillin, a semi-synthetic penicillin, is an effective antibiotic for H.
pylori infection. The frequency of amoxicillin resistant H. pylori organisms is low. The
drug rapidly accumulates in anural mucosa via systemic circulation. Its antimicrobial
activity against H. pylori depends on the pH level; the minimal inhibitory concentration
(MIC) decreases as the pH increases. (186).
The highest prevalence of sensitivity detected was against Clavunlinated Amoxicillin
which agreed with the study done in the Gemelli hospital of Rome that showed the
highest sensitivity rate in Amoxicillin (187) and also with the study done in Japan
(179) and it was higher than that of study done in United states which found the
resistance rate to Amoxicillin 1.4% (188).Data collected in 1998- 2002 found the
reissuance rate was 9 %(189). It appears that Amoxicillin resistance have remained
relitatively stable .and highest from the study done in India which found that 0.9% of
isolates were resistant to Amoxicillin (190).Reports of resistance to amoxicillin have
been extremely rare.(191,192).
Tetracycline
Helicobacter pylori resistance to Tetracycline was not reported until 1996., when
Midolo and colleagues reported Tetracycline resistant strains from Australia (193,194),
and the studies done in Italy 1997 (193,195 ) that found 8% of strain were Tetracycline
resistant
In this study the prevalence of H . pylori susceptible strain were 61.7% this high
sensitivity is slightly higher than that study done in Ninth people`s Hospital of
Shanghai that were found the sensitivity was 58% (193) and from the study done in
Douala General Hospital, Cameroon which found that Tetracycline susceptibility rates
55
were 56% (196). This prevalence of high H . pylori susceptibility rate were lowest from
the rate in study done in India which was found all the isolates were sensitive to
Tetracycline (190)..
Antibiotic treatment of H. pylori infection is not without risk. Antibiotic therapy can
lead to the development of pseudomembranous colitis, a potentially severe infection
caused by Clostridium difficile. In addition, antibiotics frequently enable the
overgrowth of Candida albicans, which can result in vaginitis, gastrointestinal
disturbances, or other complaints. Moreover, antibiotic treatment could lead to the
overgrowth of antibiotic-resistant strains of H. pylori, making further attempts at
eradication more difficult. (32).
Investigations into plant materials as alternative sources of antimicrobials have become
more common over the past few years, due to the increased rate of development of
antibiotic resistant organisms. New strategies to combat infection are also being sought
and one such strategy is the use of ‘anti-adhesive’ molecules, targeting the primary step
of infection - adhesion of the organism to host tissue (197,198).
In this study, H. pylori showed high sensitivity rate to extract of Commiphora myrrh
which were 100% in dilution one ,90% for dilution two ,60%dilution three and 20% for
dilution four that was shown as the MIC at dilution one1/20.
This high sensitivity was similar to other studies done on other bacteria and
microorganisms . The only one study done on effect on gastric ulcers done by Al-Harbi
and et al (1994) in rats who was investigated the extract of myrrh for its ability to
protect the stomach lining against laboratory-induced ulcers in rodents. They found
that pre-treatment with myrrh resulted in a dose-dependent protection against
ulceration (199).
56
Conclusion
The high rate of resistance in our patients would suggest that sensitivity testing to
alternative treatment against Helicobacter pylori is necessary.
Commiphora myrrh has wide-spectrum antimicrobial activity and it can investigated as
alternative treatment against Helicobacter pylori.
57
Recommendation
Due to the high rate of resistance to available anti-infective agents, Commiphora
myrrh can be examined as an alternative treatment on H. pylori.
58
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