Comparative of different diagnostic methods Iraqi J Pharm Sci, Vol.19(1) 2010 Helicobacter pylori 74 Study the Prevalence of Helicobacter pylori Infection by Different Diagnostic Methods Ibtihal N. Saeed* ,1 and Aseel I. Ibrahim* * Department of Clinical Laboratory Science,College of Pharmacy,University of Baghdad, Baghdad, Iraq. Abstract A total of 41 patients with gastro duodenal symptoms (show signs of inflammation with or without duodenal ulcer) . 21 males (51.2%) and 20 female (48.8%) with an average age 0f (20 – 80) years old under going gastrointestinal endoscopy at Baghdad teaching hospital in internal disease clinical laboratory , between (February – June) 2009 . Biopsies specimen of antrum , gastric fundus ,& duodenal bulb were examined by the following methods (rapid urease test , Giemsa stain section to detect bacteria , & Haematoxilin and Eosin stained section for pathological study which are considered the gold standard methods , sera or plasma from these patients were tested by immunochromotography (ICM),serological method for IgG antibodies to H. pylori. History picture are( use of certain medication , tobacco , alcohol, and current infection are taken). The results showed that the percentage of prevalence (positive results)were (83%) by histopathological method while it gave only(73%) by serological method and(66%) by rapid urease test, and the prevalence in males was more than in females (44%), (39%)respectively ,and also the prevalence increase with age (40 – 60) 14 out of 15, most patients show gastritis and duodenal ulcer, 25 (60%) by endoscopy diagnosis and 7 (17%) show malignant cancer ,while 9(22%) without any symptoms. The sensitivity of urease test (82%) and specificity (88.1%) and by ICM sensitivity (86%) and specificity (67%) comparing with gold standard methods 100% . The aim of this study is to compare the different diagnostic techniques of Helicobacter pylori infection by using invasive methods (histological examination of gastric & duodenal biopsies stained by Giemsa &Haematoxilin & Eosin methods , & rapid urease test which is considered the gold standard methods & non-invasive serological methods such as ICM rapid test , all these tests provide information about the incidence and prevalence of H. pylori in population , diagnostic value for each test also the eradication of person. Keywords: Serology, Helicobacter pylori , gastric ulcer , Diagnosis الخالصة انًسببت نمشحت انًعذة ٔاالرُى عششي فً عٍُت عشٕائٍت يٍ Helicobacter pylori ٌخضًٍ انبحذ دساست اَخشاس بكخشٌا انًشضى انزٌٍ ادخهٕا إنى شعبت انُاظٕس فً يسخشفى يذٌُت انطب انخعهًًٍ فً بغذاد ٔرنك باسخخذاو طشق حشخٍصٍت يخخهفت يُٓا ذاو صبغت انًٍٓاحٕكسٍهٍٍ ٔصبغت انكًضا ٔرنك نذساست انخغٍشاث انُسٍجٍت ٔفحص حٕاجذ انبكخشٌا فً انُسٍج , انفحص انُسٍجً باسخخ كزنك فحص انعٍُاث بطشٌمت انخحمك يٍ إَخاج إَضٌى انٍٕسٌاص باالعخًاد عهى ٔسظ انٍٕسٌا انسائم انحأي عهى دنٍم انفٍُٕل األحًش , ٔلذ ٔجذ اٌ َسبت ICMباالعخًاد عهى انطشٌمت انسشٌعت IgGٍش ٔنٕجً نألجساو انًضادة ٔكزنك بانطشٌمت انغٍش يباششة بانفحص انس . %93َسبٍا أعهى يٍ اإلَاد %::سُت ٔاٌ اإلصابت بانشجال 6>-;:االَخشاس ٔاإلصابت حضداد يع حمذو انعًش ٔخاصت بٍٍ األعًاس بًٍُا انًصابٍٍ بسشطاٌ %6>انًعذة ٔاالرُى عششي ْى حٕانً اٌ انخشخٍص األٔنً بانُاظٕس ٌبٍٍ اَّ انًشضى انًصابٍٍ بمشحت بانطشٌمت %19بًٍُا كاَج %39ٔاٌ اعهى َسبت نالَخشاس شخصج بطشٌمت انذساست انُسٍجٍت %71ٔانًشضى انعادٌٍٍ %88انًعذة بانذساست انُسٍجٍت ٔ %766عٍ طشٌك فحص اَضٌى انٍٕسٌاص , ايا كفاءة ٔحساسٍت ْزِ انطشق فخخشأح بٍٍ %>>انسٍشٔنٕجٍت ٔ .بانطشق انسٍشٔنٕجٍت ٔاالَضًٌٍت عهى انخٕانٍض 38%, %>3 Introduction In 1983 Warren and Marshal (1) isolated a new curved gram negative bacillus from gastric mucosa of patients with active chronic gastritis , this bacteria was first named Campylobacter pyloris then C. pylori and finally Helicobacter pylori (2) , establishing an association between the bacteria , gastritis and peptic ulcer disease . H. pylori is the most important cause of chronic gastritis (3,4,5 ) , it is also the most important etiological factor responsible for duodenal ulcer (3,4,5) ,gastric ulcer (3,4,5) , and has an important role in the pathogenesis of gastric cancer (6,8 ) . H. pylori is also responsible for dyspeptic patients, and screening for H. pylori in those patients improve selectivity for gastroscopy (5) . The identified virulence factors of H. pylori include the flagella used for motility through the mucus , the urease activity used for neutralizing the acid from the stomach . The cytotoxin activity which vascuolize the epithelial cells (10,11) and this examined by histopathological study . Since Marshal and Warren established the association between H. pylori , gastritis ,& peptic ulcer, a great number of diagnostic techniques have been developed (12) .The first rapid and simple test developed for the diagnosis of H. pylori infection was urease test based on the capacity of the organism to produce great quantities of this enzyme (13,14,15,16) . 1Corresponding author E- mail : Ibtihal_noori@yahoo.com Received : 15/2/2010 Accepted : 26/5/2010 Iraqi J Pharm Sci, Vol.19(1) 2010 Helicobacter pylori 75 The urease catalyzes the degradation of urea to ammonia and bicarbonate. This reaction produces an increase in the pH of the medium that can be detected by an acid – base indicator such as phenol red , that changes color from yellow to pink (15) .The velocity of the change of color depends on the urease concentration according to the numbers of bacteria present (17) .The great advantage of the urease test in the diagnosis of H. pylori is that the result can be obtained before the patient leaves the endoscopy room. The result were comparable in sensitivity and specificity with the histological and culture techniques and staining section by Giemsa stain which are considered the gold standard methods (gastric biopsy is required to perform the test) (18,19) . McNulty and Wise (15) were the first ones to use this test to detect H. pylori infection . Serological tests are useful in H. pylori infection because virtually all patients colonized with this organism under a local antibody response directed against antigens covering the surface and flagella of the organism and this antibody response detected in the serum (23,24,25) . Also serological methods used to diagnose H. pylori in which no upper gastrointestinal endoscopy is required . Maastrich 1996 working group cited by Anon suggested that screening dyspeptic patient under 45 years of age for H. pylori might reduce the need of endoscopy (20,21) , but blood must be obtained to detect H. pylori antibodies (22) .H. pylori serology is alterative in comparison with other methods because it is simple , inexpensive , & less of a burden for the patient .Several kits for detection H. pylori by serology have become commercially available since the discovery of H. pylori by Warren in 1983 (1) , most of these kits are based on various antibody preparations and different techniques , this lead to an increase in the number of studies that have evaluated kit characteristics . Different studies for comparison between kits to account for the different reference standards and designs used by various investigators (22,23,24,25) . Serological diagnosis simplest and least expensive , non – invasive method for IgG and or IgA antibodies , latex agglutination methods are quick tests , useful for screening purposes .ELISA based tests accurately quantities the amount of antibody (titer) present and are promising tool for assessing the efficacy of H. pylori eradication treatment (27) , also for rapid office – based serologic test , using immunochromotography (ICM) , and the immunoblott for the diagnosis of H. pylori (26) . C13 / C 14 , urea breath test are reliable non – invasive methods for diagnosis of on going H. pylori infection (30,31) . Material and methods Samples : 41 gastric and duodenal biopsies from patients of the endoscopy department of Baghdad teaching hospital – Baghdad / Iraq , were analyzed between (February – June) 2009 , at least two biopsies were taken from the antrum of each patient for histological study send to histopathological laboratory of the hospital stained by Giemza method (Luna 1968) (32) & Haematoxilin & Eosin method (Modified m. of Guyer ,1953 ) (33) by (Gram Wegurt) to study the histological change and detecting rod shaped H. pylori. Phenol red rapid urease test A solution of urea 10% and solution of phenol red 1% were prepared for the working solution , 0.1ml of phenol red solution were mixed in 1 ml of the urea solution . The reagent is stable for two weeks of 4 – 8 °C each biopsy was embedded in 0.2 ml of the reagent and incubated at room temperature (22°C) for 1 min. Serological diagnosis by (ICM) immunochromotography method of (ACON H. pylori one step –rapid test Devise) Serum / plasma is a sample test that utilized a combination of H. pylori antigen coated particles and anti – human IgG to qualitatively and selectively detect H. pylori antibodies in serum or plasma in10 minutes after serum or plasma specimen is placed in the specimen well., it reads with H. pylori antigen coated particles in the test .The mixture migrate chromatographically along the length of the test strip and interacts with the immobilized anti – human IgG , if the specimen contain H. pylori antibodies , a colored line appear in the test line region , indicating a positive result , if the specimen dose not contain H. pylori antibodies a colored line will not appear in this region , indicating a negative result comparing with positive control – test , the result should be read at 10 min.( ACON lab. Inc – 4/08 Sorrento Valley Boulevard .San Diego ,CA 9212,USA). Personal information about past infection , treated use of certain medication , alcohol and tobacco , this result were analyzed according to age , sex ,race and another characteristics . Calculation of sensitivity and specificity Positive and negative predictive values were made using the following formula : Iraqi J Pharm Sci, Vol.19(1) 2010 Helicobacter pylori 76 Results A total of 41 patients were investigated, 21(51.2%) male and 20(48.8%) female with mean age 45 years old (range 20-80year), these patients under examination showed by endoscopy diagnosis that 14(34%) of them have gastric ulcer, 11(27%) duodenal ulcer, 7(17%) with gastric cancer and 9(22%) non ulcer dyspepsia. Tables 1&2 show that the percentage of infection with Helicobacter pylori or the prevalence of infection which studed by histopathological and Giemsa staining section methods increase in males 18(44%) more than in females 16(39%) and also the percentage of infections increase with age between (40-60) years old 14(34%) out of 15(36.5%) patients, the percentage of infections more than in younger and older patients. Table (3) shows the relation between the endoscopy diagnosis with positive and negative result of infection done by different diagnostic methods , in which histopathology and Giemsa staining methods gives 34(83.0%) positive, 7(17%) negative, by serodaignosis (ICM) test give 30(73%) positive, 11(27%) patients negative while by rapid urease test 27(66%) positive, 14(34%) patients negative. The positive value of serodignosis and urease test consist 88%,79% respectively from the true positive value by histopathological study (34+) patients.From these result the high prevalence of infections were obtained first by histopathological study then by serodiagnosis methods and later the lowest value by urease test. In all test used the prevalence over 75% considered high prevalence of infection in population (6) . Also if we determined the positive value of diagnosis in relation with disease or endoscopy finding, histopathological study gives 90% positive in duodenal ulcer and 80% with gastric ulcer comparing with serodiagnosis (82%),(72%) and urease test (72%),(54%) irrespectively. Table (5) show the sensitivity& specificity for each test depending on true positive, true negative, false positive, false negative values determined in table (4), comparing with a gold standard method of diagnosis and this gives the sensitivity & specificity of histopathology & Giemsa staining methods 100% , by serodiagnosis (ICM) method (86%), (67%) and by urease broth test (82%), (87%), means that the first method gives more accuracy result than others, with many disadvantage, and the other methods give less accuracy with many advantages discussed later in discussion. Table 1 : Show the prevalence of H. pylori infection in male & female. Percentage of infection Number of positive H. pylori Percentage Total number 44% 18 51.3% Male (21) 39% 16 48.7% Female (20) Table 2 : Show the prevalence of infection in different age groups. Result (%) Number of positive Percentage (%) Total number (41) Age in years 14.6 6 21.95 9 20 – 30 12.5 9 29.2 12 31 – 40 17.0 7 17 7 41 – 50 17.0 7 19.5 8 51 – 60 7.3 3 7.3 3 61 – 70 4.8 2 4.8 2 71 - 80 Iraqi J Pharm Sci, Vol.19(1) 2010 Helicobacter pylori 77 Table 3 : Show the percentage of positive and negative H. pylori infection diagnosed by urease , serological kit and endoscopy biopsies. Sum. Non- Ulcer Dyspepsia No.(%) Gastric cancer No. (%) Duodenal ulcer No.(%) Gastric ulcer No.(%) Total number (41) 41(100%) 34(83%) 30(73%) 27(66%) 9 (22%) 6(14.63) 7(17%) 5 (12.19) 7 (17%) 7(17%) 4(9.75%) 5 (12.19) 11 (26 %) 10(24.4%) 9(21.95%) 8 (19.5%) 14 (34%) 11(26.8%) 10(24.4%) 9(21.95%) Histopatholog Giemsa stain Sero.Kit Urease H. pylori (+) 7(17%) 11(27%) 14(34%) 3(7.3%) 2 (4.8% ) 4 (9.75 ) 0 3 (7.31%) 2 (4.87% ) 1(2.43%) 2 (4.87%) 3 (7.3%) 3(7.3%) 4 (27%) 5 (12.2% ) Histopatholog Giemsa stain Sero. kit Urease . H pylori (-) Table 4 : Number of true positive , true negative ,false positive ,false negative by different diagnostic methods. Table 5 : values of different diagnostic methods . N.P.V. % P.P.V. % Specificity % Sensitivity % Test 100 100 100 100 Histopath. 100 100 100 100 Giemsa stain 54 96 88 82 Rapid Urease test 45 94 67 86 Rapid serodiagnosis (ICM) If you know the prevalence of Helicobacter pylori in your population you can make a judgment about the predictive value of a positive or negative test from the table(6). Table 6 : Predictive value of a test with 85% sensitivity and 79% specificity Probability of disease with negative result(%) Probability of disease with positive result(%) Prevalence of disease 2 31 10 16 80 50 63 97 90 Test True positive False positive True negative False negative Histological changes 34 0 7 0 Geimza stain 34 0 7 0 Urease 27 1 7 6 ICM (Kit) serology 30 2 4 5 Iraqi J Pharm Sci, Vol.19(1) 2010 Helicobacter pylori 78 Discussion The aim of this study is to determine the prevalence of infection with H. pylori by different diagnostic methods in random population enter the endoscopy department for gastric and duodenal examination with or without symptoms of inflammation. From the 41 patients under investigations results in table (1) showed that prevalence of infection in males more than in females and these results agree with other studies done by Nicholas et al (1992), in which they found that 47% males out of 82 patients, (44%) 0f them were infected by H. pylori (25) . Table (2) showed that prevalence of infection increase with age between (40-60) years old agreed with Nulty (1999) which found that the more likely age of infection in patients over 50 years old (42%) than in younger patients (21%) (22) , another group of Liston R, et al (1996) cited by Nulty(1999), found that (31.7%) of elderly patients with seropositive result had no evidence of active infection determined by endoscopy and urease test. Older patients are more likely to have developed atrophic gastritis and H. pylori can not readily colonize this type of gastric mucosa ( 22) . It was recognized that prevalence of H. pylori infection increase with age in a symptomatic persons in developed countries and this tend to plateau at around the age of 60 years, related to socioeconomic status and ethnicity (5,23,25) .Table(3)which showed high prevalence of infection given by histopathological study which are considered invasive gold standard methods (22,25,26) , (83%) of patients gave positive result ,while by serodiagnosis (ICM) method (73%) and (65%) by urease test method which means that these methods gives lowest value of prevalence comparing with histopathological study and this because many factors were affecting on the value of the result such as for example. - Negative values in biopsy methods histopathology & Giemsa staining section depend on patient that may be under treatment or past proven H. pylori infection treated with a course of antimicrobial drugs with proton pump inhibitors , that patient give negative and clearance of the disease in biopsy specimen but can give positive result with serodiagnosis, and so give false positive result and high prevalence than biopsies (23,25) . - A negative value in urease test depend on non homogeneous distribution of the microorganism in the stomach and this situation is overcomed by use of several specimen from (3-5) for the same patient (34,35,36) so we minimize the specimen error and this explain the 13 patients which give negative result by this method, which lowering the percentage of infection comparing with other methods. -In serodiagnostic method the percentage of infection (73%) which gives positive result and 11(27%) patients comparing with histopathological study , 4 patients only were true negative and 5 patients were false negative and only 2 patients gave false positive result , this can be explained by:  Patients who are in acute case of infection before an IgG response has developed gave false negative serological result ,means that it may be positive result in biopsy method, also negative result may be due to patient not produce circulating antibody response which detectable only with complex type of antigen (Preez-Preez, et al. cited by Nicholas(1992) (25) .  False positive result according to cross reaction antigen (3-9%) of patient have false positive result with H. pylori that might produce antibodies such as Gastrospirillum hominis and this also depend on type of antigen used in test (23,25) or it may be that patient with past infection that gives false positive result with slowly return antibody, it may give positive test for over 6 months from clearance of the disease (25,26,34) . Table (5)show the sensitivity & specificity of each test depend on the true positive ,true negative , false positive ,& false negative value in table (4), each positive value in histopathological study considered true positive value and each negative value considered true negative value (gold standard methods) and so sensitivity & specificity (100%) and this also agreed with other study which find that these methods gives sensitivity & specificity between (98-100%) (34) , and for serodiagnosis (86% ) , ( 67%) while urease test gives sensitivity & specificity (82%),(87%).The sensitivity & specificity of serological test reported by many workers varies from (76-96%) and half of the patients with false positive result were over 50 years age. Other group found that elderly patient with positive serology had no evidence of active infection determined by endoscopy and urea breath test (14,22) , other workers for the same methods (ICM) test find that the sensitivity is (96%) with(94%) specificity (26) which used this test to diagnose H. pylori Iraqi J Pharm Sci, Vol.19(1) 2010 Helicobacter pylori 79 infections in thai children between(1.5-16 years old), other study compare different serological kits for H. pylori infection and also found that the sensitivity and specificity around (67%-86%) (22) , other outhers (25) find that sensitivity of commercial ELISA kits had an accuracy between (89%- 95%).The sensitivity of urease test according to Eugenia (34) is record to be (97%) by phenol red broth test with (100%) specificity also he mention that other authors have reported that urease test with specificity between (98%-100%) and sensitivity between(64%-98%),and this agreed by Vaira (37,38) , Thillainayagam (39) , Mal.fertheiner (40) , McNulty (15) , Arvid, Morris (42) , &Westblom (14) reports specificities of (86%,98%,92%,100%) and sensitivities of (84%,92%,100%,88%) respectively, only Hernandez reports a sensitivity of (72%) and specificity of (83%) for Christensen's urea broth (43) .The presence of false negative and false positive result may be explained by the patchy distribution that H. pylori present in gastric mucosa ,especially in the body and fundus of the stomach, so the microorganism can be present in one biopsy and absent in another from the same patient (34, 44,45) . So the false negative value by this test were caused by the patchy distribution of the bacteria. Conclusion It has been proposal that patient with dyspepsia could be screened for H. pylori status before it is recommended (25,22,23) and as H. pylori occurs in over (90%) of patient with chronic duodenal ulceration and up to (80%) of patient with chronic gastric ulceration (25,16,21) , it has been proposal that such an approach would help to reduce the need for endoscopy as well as cost, if such a policy were adopted only seropositive patient would undergo endoscopy and over 45 years of age or those taking anti inflammatory drugs, this would avoid up to (23%) of endoscopies. However ,further large prospective clinical studies are needed before such an approach can be accepted. Also serological methods can be used for monitoring treatment and successful eradication of infection by detecting the fall in level of IgG antibodies in serum after 3 months of treatment. The great advantage of the urease test in the diagnosis of H. pylori is that the result can be obtained before the patient leaves the endoscopy room, making clinical management easier. The studies suggest that urease result comparable in sensitivity and specificity with histological and culture techniques, being more economic and faster (12,34) , Nevertheless an endoscopy is always necessary because a gastric biopsy is required to perform the test and also culture can be required for evaluating the sensitivity to antibiotics, so urease test should be done jointly with another diagnostic test as histology or culture. Some authors and reports go to connect in a table between prevalence and sensitivity & specificity of different methods (Table6). References 1. Warren ,J.R., and Marshall .1983. Unidentified curved bacilli on gastric epithelium in active chronic gastritis . Lancet ii :1273- 1275. (letter.) . 2. Hernandez F., Rivera P., Sigaran , Aguilar M., Miranda J., Rodriguez D. , et al. ,1990. The first cases of Helicobacter pylori (Campylobacter pylori) reported from costa Rica . Rev. Biol. Trop. 38 : 481-2. 3. Anderson ,L., S. Holck, C. Povlsen, L. , Elsborg and T. justesen. 1987. Campylobacter pyloridis in peptic ulcer disease .Scand. J. Gastroenterol. 22 : 219-224. 4. Buck, G.E. 1990. Campylobacter pylori and gastroduodenal disease . Clin. Microbiol. Rev. 3 : 1-12. 5. Dooley C. P., H. Cohen ,P.L., Fitz gibbons , et al. 1989. prevalence of Helicobacter pylori infection and histological gastritis in a symptomatic persons.N.Engl. J. Med. 321 :1562-1566. 6. Muller, L.B.,Fagundes, R.B., Moraes, C.C., Rampazzo, A.2007. Prevalence of Helicobacter pylori infection and gastric cancer precursor lesions in patients with dyspepsia .Arg. Gastroenterol. Apr. Jun ; 44:93-8. 7. Jaskiewicz, K., H.D. Ouwrens, C.W. Woodruff, M.J. Van Wik, and S.K. Pricee. 1989. The association of Campylobacter pylori with mucosal pathological changes in population at risk for gastric cancer. S.Afr. Med.J. 75:417- 419. 8. Tatsuta M., Tishi H., Okuda S., Taniguchi H., Vokota Y.1993. The association of Helicobacter pylori with differentiate type gastric cancer .Cancer ; 72 :1841-5. 9. Hu. P. Mitchell H., Li. Y.,Znou M.,Hazell S.1994.Association of Helicobacter pylori with gastric cancer and observations on the detection of this bacterium in gastric cancer cases. Am.J. Gastroenterol. ; 89:1806-10. 10. Clyne M.,Labigne A., Drumm B.1995. Helicobacter pylori requires and acidic Iraqi J Pharm Sci, Vol.19(1) 2010 Helicobacter pylori 80 environment to survive 4 n the presence of urea. Infect. Immunol.; 63: 1669-73. 11. Xiang Z., Censini S., Bayeli P., Telford J., Figura N., Rappuoli R., et al.1995. Analysis of expression of Cag A and Vac A virulence factors in 43 strains of Helicobacter pylori reveals that clinical isolates can be divided in to two major types and Cag A is not necessary for expression of the vacuolating cytotoxin.Infect. Immunol. ;36:49-8. 12. Glupczynski Y.,Diagnostico microbiologico de la infection por H. pylori . En lopez-Brea .Med.1995. Helicobacter pylori. Microbiologia ,clinica Y tratamiento. Madrid :Ed. Mosby / Doyma Libros; P. 241-59. 13. Langenberg M.,Tytgat G., Schipper M.1984. Campylobacter like- organisms in stomach of patients and healthy individuals. Lancet . ;I:1348-49. 14. Westblom T.,Madan E., Kemp J., Subik M.1988. Evaluation of rapid urease test to detect Campylobacter pylori infection .J. Clin. Microbiol .; 26:1393-4. 15. McNulty C., Wise R.1985. Rapid diagnosis of Campylobacter associated gastritis . Lancet ;I:1443-4. 16. 16Hemandez F., Rivera P., Sigaran M., Miranda J.1991.Diagnosis of Helicobacter pylori comparison of an urease test, histological visualization of curved bacteria and culture. Rev. Inst. Med.Trop Sao Pablo ; 33: 80-2. 17. Lamouliatte H., Cayla R.,Daskalopoulos G. Upper digestive tract endoscopy and rapid diagnosis of Helicobacter pylori infection .En Lee A. and Megraud F.Helicobacter pylori : Techniques for clinical diagnosis and basic research .London :W.B Saunders. 18. Goodwin CS., Blincow EW., Warren JR., Waters TE., Sanderson CR. Easton L .1985 . Evaluation of culture techniques for isolation Campylobacter pyloridis from endoscopy biopsies of gastric mucosa.J.Clin.Pathol.;38: 1127-31. 19. Gray SF., Wyatt JT., Rathbone BJ.1986. Simplified techniques for identifying Campylobacter pyloridis.J.Clin.Pathol. :39:1279. 20. Patel P., Khulusi S., Mendell MA., Lioyd R., Jazrawi R., Maxwell TD., et al.1995. Prospective screening of dyspeptic patients by Helicobacter pylori serology .Lancet;346: 1315-8. 21. Anon. Current European concepts in the management of Helicobacter pylori infection. The Maastrich Consensus Report .Gut;41: 8-13. 22. CAM MucNulty,P. Nair, BE. Watson, Jsuff, RMValori.1999.A comparison of six commercial kits for Helicobacter pylori detection. Communicable disease and public Health. 23. Evans, D.J.,D.G. Evans, D.Y. Graham, and P.D. Klein.1989.A sensitivity and specific serologic test for detection of Campylobacter pylori infection. Gastroenterology 96: 1004-1008. 24. Marshall, B.J., D. B. McGechle, G.J. Francis nd P.J. Utley. 1984. Pylori Campylobacter. Serology . Lancetii :281 . (Letter to the editor). 25. Talley, J.N., Kost, L.,Haddad,A., and Zinsmeister, R.A. 1992 . Comparison of commercial serological tests for detection of Helicobacter pylori antibodies. Journal of Clin.Microbiol.Des.Vol.30,No. 12 , 3146-3150. 26. Treepongkaruna, S., Nopchinda, S., Taweewongsoun, A.A rapid serologic test and immunoblotting for the detection of H. pylori infection in children.Journal of tropical pediatrics ,Oxford Journals. Medicin, Vol. 52, No. 4, PP:267-271. 27. LE. Crabtree,TM. Shallcross, RV. Heatley, and JI. Wyatt. 1991. Evaluation of commercial ELISA for serodiagnosis of Helicobacter pylori infection. Jour.of Clini. Path.;44:326-328. 28. Yukio bata, Shogo Kikuchi, Hiroto Miwa, Kiyoko Yagyu, Yingsong Lin, and Atsushi Ogihara. Diagnostic accuracy of serological kits for Helicobacter pylori infection with the same assay system but different antigens in a Japanese patient population. 29. R.J.F. Lahiej, H. Straatman, J.B.M.J. Jansen, and A.L.M. Verbeek. 1998. Evaluation of commercially available H. pylori serology kits: a Review. Journal of Clinic. Microbiology,October, Vol. 36, No. 10,P:2803-2809. 30. Bell GD, Harrison G, Morden A, Jones PH, Gant PW, et al. 1987. C 13 -urea breath analysis, a non-invasive test for Campylobacter pylori in the stomach. Lancet; I: 1367-8. 31. Rauws EAJ, Van Royens EA, Langenberg W, Woensel JV, Trij AA, Tytgat GN. 1989. C 14 -urea breath test in C. pylori gastritis. Gut ;30:798-803. 32. Luna, L.G.,1968.Manual of histological staining methods of armed Forces institute of pathology 3th edition.The black stone Iraqi J Pharm Sci, Vol.19(1) 2010 Helicobacter pylori 81 division,Mcgyos, Hill book Co., New York. 33. Guyer, M.F.,1953. Animal micrology 5 th edition .The university of Chicago press. Chicago. 34. Eugenia Ma.,Quintana-Guzman,Karl Schosinky-Nevermann, Maria L. Arias- Echandi, Henry Davidovich-Rose, 1999.Comparative study of urease tests for Helicobacter pylori detection In gastric biopsies.Rev. Biomed.;10:145-151. 35. Kjoller M,Fischer A,Justesen T,1992. Transport conditions and Number of biopsies necessary for culture of Helicobacter pylori,Eur. J. Clin. Microbiol. Infect. Dis.;10:166-7. 36. Van der Hulst R, Verheuls S, Weel J, Gerrits Y, Ten F, Dankert J, 1996. Effect of specimen collection techniques, transport media and Incubation of cultures on the detection rate of Helicobacter pylori ,Eur. J. Clin. Microbiol. Infect. Dis.;15:211-5. 37. Varia D, Holton J, Cairns S,1996. Urease test for Campylobacter Pylori infection. Eur. J. Gastroenterol. Hepatol;8:53-6. 38. Varia D, Holton J, Cains S, Polydorou A, Falzon M, Dowsett J, Salmon P,1988. Urease test for Campylobacter pylori :Care in Interpretation .J. Clin. Pathol.;41:812-3. 39. Thillainayagam A, Arvid S, Cook R, Harrison I, Tabaqchali S, Farthing M, 1991. Diagnostic efficiency of an ultra rapid endoscopyRoom test for Helicobacter pylori. Gut; 32:467-9. 40. Malfertheiner P, Dominguez-Munoz J, Hekenmuller H, Neubrand M, Fisher H, Sauer P. 1996. Modified rapid urease test for detection of Helicobacter pylori infection. Eur. J. Gastroenterol. Heptol.;8:53-6. 41. Arvid A, Cook R, Tabaqchali S, Farthing M.1988. One minute Endoscopy room test or Campylobacter pylori. Lancet;1:704. 42. Morris A, Mclntyre D,Rose T,Nicholson G.1986.Rapid diagnosis of Campylobacter pyloridis in infection .Lancet;1:149. 43. Hernandez F, Rivera P, Sigaran M, Miranada J.1991. Daignosis of Helicobacter pylori: Comparison of an urease test ,histological Visualization of curved bacteria and culture.Rev. Inst. Med. Trop Sao Pablo; 33:80-2. 44. Barthel J, Everett D.1990. Diagnosis of Campylobacter pylori Infections The"Gold standard" and the alternatives. Rev Infect. Dis.;12:5107-14. 45. Azuma T, Kato T, Hirai M, Ito S, Kohli Y.1996. Diagnosis of Helicobacter pylori inection. J. Gastroenterol. Hepatol; 11:662-9.