Vinay Hajare*, H. Anandkumar and R.S. Rajeshwari
The H.pylori is one of the most common infections in the world. It causes dyspepsia, chronic gastritis and gastric carcinoma. Spread of the infection from person to person is by oral-oral or faeco-oral route. The aim of the study is to determine the rate of isolation of H.pylori infection from different sites like gastric biopsy, gastric aspirate, saliva and faeces. Patients were selected based on the clinical findings like early satiation, epigastric pain, epigastric burning, bloating, belching and endoscopic findings. Four types of samples- Gastric biopsy, Gastric aspirate, Saliva and Faeces were collected and all the four samples were subjected to Direct Gram Stain, Rapid Urease Test and Culture. Culture was done by inoculating samples onto Skirrows media with cefsulodin and amphotericin B and incubated at 35ºC for 3-5 days under microaerophilic conditions. Identification of organism was done based on gram stain of the colony, oxidase, catalase and urease test. Out of 96 clinically suspected cases of gastritis, H.pylori was isolated in 34(35.41%) patients from Gastric biopsy, 18 (18.75%) patients from Gastric aspirate, 8 (8.33%) patients from saliva and 4 (4.16%) patients from faeces. Among the 96 patients selected 59 were male participants and 37 were female participants with age between 20-60 years. In the present study, we found that H.pylori can be cultured from biopsy, gastric aspirate, saliva and faeces. There are several invasive and non invasive techniques to diagnose H.pylori, each having its own advantages and disadvantages. Non-invasive tests on saliva and faeces sample can play an important role in the diagnosis of H.pylori infection, but has the disadvantage of low sensitivity, whereas invasive tests like gastric biopsy and gastric aspirate are more sensitive.
Keywords: H. pylori; Gastritis; Gastric biopsy.
The presences of spiral organisms were first reported in the stomachs of mammals over 120 year’s ago1. Marshal tried to culture the bacteria by using methods to culture Campylobacter for 48 hours but was in vain2. The first successful culture occurred when the 35th biopsy culture was left in incubator for 5 days over the Easter holidays in April 1982. Marshal proved the association of the isolated bacteria as a causative agent of gastritis. As the bacteria were closely associated with Campylobacter it was named as Campylobacter jejuni3. Campylobacter became Helicobacter when it was found that the 16s ribosomal RNA did not have characteristic sequence found in Campylobacter4.
H.pylori is a spiral shaped or curved microaerophilic Gram-negative motile bacteria equipped with 4-6 flagellae at one end. Gastric acid kills most bacteria but H.pylori has evolved some special features which allow them to live in acidic niche. Urea diffuses freely from plasma into gastric juice. The urease enzyme produced by H.pylori digests urea to produce carbon dioxide and ammonium hydroxide which neutralizes the gastric acid5-9.
H.pylori causes dyspepsia, chronic gastritis and gastric carcinoma10. Spread of the infection from person to person is by oral-oral or faeco-oral route. It has been shown that H.pylori can survive up to 3-days in water11.
The aim of the study is to determine the rate of isolation of H.pylori infection from different sites like gastric biopsy, gastric aspirate, saliva and faeces.
Materials and Methods
Informed consent mentioning that data from the diagnosis would be used for research purpose was obtained from all the patients.
Patients were selected based on the clinical findings like early satiation, epigastric pain, epigastric burning, bloating, belching and endoscopic findings.
Based on endoscopic findings, the patients were divided into four groups:
Group-1: Normal findings
Group-2: Non-ulcerative with evidence of erythema, erosion, nodularity, atrophy, plaque or petechiae
Group-4: Both of group-2 and group-3
Patients pertaining to group 2, 3 and 4 were selected for the study
Patients who are on antibiotics, Antacids, pregnancy or history of gastrectomy were excluded from the study.
Patients were instructed by the gastroenterologist not to eat or drink for eight hours before endoscopy procedure. The patients received 10% xylocaine spray as local anaesthesia in the oropharyngeal region before the procedure. The endoscope and the biopsy forceps were cleaned by savlon and then sterilized by cidex for 10 minutes. Two biopsy specimens were collected from each patient, one for direct Gram stain and other for inoculation in culture media. Four types of samples were collected from each patient. The four samples are Gastric biopsy, Gastric aspirate, Saliva and Faeces. All the four samples were subjected to Direct Gram Stain, Rapid Urease Test12, 13 and Culture.
Direct gram stain showed S-shaped bacilli 2.5 to 4 mm long and 0.5 to 1 mm thick. All the specimens were inoculated onto Skirrows media with cefsulodin and amphotericin B (Dent’s selective media) and incubated at 35ºC for 3-5 days under microaerophilic conditions.
The colonies were discrete translucent. Identification was done based on gram stain of the colony, positive oxidase, catalase and urease test12.
Based on clinical findings, a total number of 147 endoscopies were done in a period of 6 months, of which 96 (65.3%) patients were falling under group 2, 3 or 4. Among the 96 patients selected 59 were male participants and 37 were female participants with age between 20-60 years. H.pylori was isolated in 34 patients from Gastric biopsy, 18 patients from Gastric aspirate, 8 patients from saliva and 4 patients from Faeces. The isolation of H.pylori from Gastric biopsy was 35.41%, from gastric aspirate was 18.75%, from saliva 8.33% and from faeces 4.16%.
Table-1: Rate of isolation of H.pylori from different clinical samples from patients suffering from gastritis is shown in table below:
No. of Isolation of H.pylori
(n = 34)
Only Gastric biopsy positive
Gastric biopsy* and Gastric aspirate positive
Gastric biopsy and Saliva positive
Gastric biopsy and faeces positive
* Gastric biopsy is taken as gold standard specimen for H.pylori isolation
Table-2: Demographic And Clinical Characteristics of Patients Included In the study
|Variables||Total number of patients n=96||H.pylori positive
In the above study, we have observed that the sensitivity of gastric biopsy was much more compared to gastric aspirate, saliva and faeces respectively.
Oshowo A et al14 reported 17% isolation of H.pylori from gastric juice, 55% from gastric biopsy, which showed that the isolation of H.pylori from biopsy was high when compared to the results of present study, whereas the results were almost similar for gastric juice. Contrary to our work, Allaker RP et al11 reported 25% isolation of H.pylori from faeces in gastric biopsy positive patients, which was high.
Young KA15 et al reported 50% positive results from Gastric biopsy and 37% from Gastric juice and according to Momtaz H16 10.72% isolates were from saliva and 71.67% from faeces and 77.88% from gastric biopsies. These results were also in increasing trend compared to the present study.
Matsukura N et al17 showed the isolation of H.pylori in gastric aspirate to be 38% and biopsy 40%. Uribe R et al18 isolated 94.1% of H.pylori from gastric aspirate by PCR, which is more sensitive than culture.
In our study, the prevalence of H.pylori was more in males (35.9%) when compared to females (22.03%). The rate of H.pylori infection was more in males when compared to females. Similar results were shown by Khan AR et al19 where the rate of infection in males was 69% and females was 31%. He also showed that there was no significant difference between different age groups. Kanbay et al20 showed that the rate of infection increased along with the age and women were infected more commonly than men. Whereas our study showed that the rate of infection is low between 50-60 years of age group and men are more commonly infected than women.
The H.pylori is one of the most common infections in the world. It is present in 70-90% of the population in developing countries and 25–50% in developed countries21.
In the present study, we found that H.pylori can be cultured from biopsy, gastric aspirate, saliva and faeces. Helicobacter pylori were less reliably cultured from stool than from saliva. There are several invasive and non invasive techniques to diagnose H.pylori, each having its own advantages and disadvantages. Non-invasive tests on saliva and faeces sample can play an important role in the diagnosis of H.pylori infection, but has the disadvantage of low sensitivity, whereas invasive tests like gastric biopsy and gastric aspirate are more sensitive.
- Bottcher G: Dorpater Med z 1875; 184.
- Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984; i: 1311-1315.
- Warren JR, Marshall BJ. Unidentified curved bacillus on the gastric epithelium in active chronic gastritis. Lancet 1983; 1: 1273-75.
- Goodwin CS, Armstrong JA, Chilvers T et al. Transfer of Campylobacter pylori and Campylobacter mustalae to Helicobacter gen. nov. and Helicobacter pylori comb. nov. and Helicobacter Mustelae comb. nov. respectively. Int J Syst Bacteriol. 1989; 39: 397-405.
- Hu LT, Mobley HL. Expression of catalytically active recombinant Helicobacter pylori urease at wild-type levels in Escherichia coli. Infect Immun 1993; 61: 2563-2569.
- Marshall BJ, Barrett LJ, Prakash C et al. Urea protects Helicobacter (Campylobacter) pylori from the bactericidal effect of acid. Gastroenterology 1990; 99: 697-702.
- Eaton KA, Brooks CL, Morgan DR, Krakowka S. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun 1991; 59: 2470-2475.
- Butcher GP, Ryder SD, Hughes SJ et al. Use of an ammonia electrode for rapid quantification of Helicobacter pylori urease: its use in the endoscopy room and in the assessment of urease inhibition by bismuth subsalicylate. Digestion 1992; 53: 142-148.
- Nagata K, Satoh H, Iwahi T et al. Potent inhibitory action of the gastric proton pump inhibitor lansoprazole against urease activity of Helicobacter pylori: unique action selective for H.pylori cells. Antimicrob Agenst Chemother 1993; 37: 769-774.
- Zhang W, Lu H, Graham DY. An update on Helicobacter pylori as the cause of gastric cancer. Gastrointest Tumors. 2014; 1(3): 155-65.
- Allaker RP, Young KA, Hardie JM. Prevalence of Helicobacter pylori at oral and gastrointestinal sites in children: Evidence for possible oral to oral transmission. J Med Microbiol 2002; 51(4): 312-17.
- Old DC. Vibrio, Aeromonas, Plesiomonas, Campylobacter, Acrobacter, Helicobacter, Wolinella. In: Collec JG, Fraser Ag, Marmian BP, Sommons A, Mackie and McCartney’s Practical Medical Microbiology. 14th, Churchill Livingstone, 1996; 425-28.
- Tseng CA, Wang WM, Wu DC. Comparison of the clinical feasibility of three rapid urease tests in the diagnosis of Helicobacter pylori infection. Dig Dis Sci. 2005; 50: 49-52.
- Oshowo A, Gillam D, Botha A, Tunio M, Holton S, Boulos P, Hobsley M. Ann Periodontol. 1998 Supp; 3(1): 276-80.
- Young KA, Akyon Y, Rampton DS, Barton SG, Allaker RP, Hardie JM, Feldman RA. J Med Microbiol. 2000 Apr; 49(4): .343-7.
- Momtaz H, Souod N, Dabiri H, Sarshar M. World J Gastroenterology 2012 May 7; 18(17): 2105-11. doi: 10.3748/ wjg.v18.ii7.2015.
- Matsukura N, Onda M, Tokumag A, Kato S, Yamashita K, Ohbayashi M. J Gastroenterol. 1995 Dec; 30(6): 689-95.
- Uribe R, Fujioka T, Ito A, Nishizono A, Nasu M. Kansenshogaku Zasshi. 1998 Feb; 72(2): 114-22.
- Khan AR. Ann Saudi Med. 1998 Jan-Feb;18(1):6-8.
- Kanbay M, Gür G, Arslan H, Yilmaz U, Boyacioglu S. Dig Dis Sci. 2005 Jul;50(7):1214-7.
- Kabir S. Detection of helicobacter pylori in faeces by culture, PCR and enzyme immunoassay. J Med Microbiol. 2001;50:1021-29.