|Year : 2003 | Volume
| Issue : 1 | Page : 46-48
Comparative evaluation of conventional methods and elisa based IgG antibodies detection for diagnosis of helicobacter pylori infection in cases of dyspepsia
U Arora , A Aggarwal , K Singh
Department of Microbiology, Government Medical College, Amritsar, Punjab, India
Department of Microbiology, Government Medical College, Amritsar, Punjab, India
Seventy five gastric biopsy specimens and 75 serum samples of same patients complaining of dyspepsia were collected. Biopsy specimens were processed for rapid urease test, gram staining and culture. Serum samples were used for detecting IgG antibodies against 128kDa external protein (Cog A) of H.pylori using a commercially available ELISA kit. Rapid urease test was positive in 54 (72%), culture in 21 (28%) and gram staining in 15 (20%). Significant IgG levels were detected in 57 (76%) cases. It was therefore concluded that for diagnosis of H.pylori infection in cases of dyspepsia, determination of IgG levels can act as an important screening procedure.
|How to cite this article:|
Arora U, Aggarwal A, Singh K. Comparative evaluation of conventional methods and elisa based IgG antibodies detection for diagnosis of helicobacter pylori infection in cases of dyspepsia. Indian J Med Microbiol 2003;21:46-8
|How to cite this URL:|
Arora U, Aggarwal A, Singh K. Comparative evaluation of conventional methods and elisa based IgG antibodies detection for diagnosis of helicobacter pylori infection in cases of dyspepsia. Indian J Med Microbiol [serial online] 2003 [cited 2019 Dec 13];21:46-8. Available from: http://www.ijmm.org/text.asp?2003/21/1/46/8317
The discovery of Helicobacter pylori en and Marshall not only introduced a whole new group of bacteria to science but also revolutionized our concept of gastroduodenal pathology and diverted the world wide attention from pH to Hp. Presently, its role has been established in chronic antral gastritis, duodenal ulcer, chronic gastric ulcer, dyspepsia, gastric cancer and gastric lymphoma. World Health Organization added H.pylori to its list of known carcinogens. The diagnosis of H.pylori infection is currently based upon endoscopic biopsy based tests (Rapid urease, culture, Gram staining and histopathology.
These procedures are invasive, tedious and hence every person complaining of symptoms suggestive of gastroduodenal disease cannot be subjected to these tests. Non invasive, serology based tests detecting IgG antibodies (since H.pylori causes chronic infection) are commercially available. The present study was therefore planned for comparative evaluation of conventional methods and ELISA based IgG antibody detection by a commercially available kit for diagnosis of H.pylori infection in cases of dyspepsia.
| ~ Materials and Methods|| |
Seventy five patients attending the OPD of Guru Nanak Dev Hospital, Amritsar for dyspepsia (ulcer and non ulcer type) were subjected to endoscopy at Endoscopy Centre, Amritsar.
Collection of specimens
Blood: 5 mL of venous blood of patients was collected, serum was separated and stored at -20°C for further processing.
Antral biopsy: Two pieces of antral biopsy were taken, one was inoculated in rapid urease test and another in normal saline. The time was noted and specimens were immediately transported to the laboratory.
Processing of specimens
Rapid urease test
0.5 mL of 10% urea with 0.002% phenol red at pH 6.5 was used and observed for 30 minutes after inoculation.
Biopsy specimens were crushed in a sterile glass tissue homogenizer and a smear was prepared on clean glass slides from the homogenate, fixed by methanol and stained by Gram staining.
Tissue homogenate was inoculated on brain heart infusion agar supplemented with 7% sheep blood with and without antibiotics (vancomycin 6 mg, polymyxin B 2500 units and amphotericin B 3 mg per liter). The plates were incubated at 37°C in a candle jar with a pad of cotton soaked in water placed at the bottom. The plates were examined on 3rd, 5th and 7th day of incubation. Characteristic colonies were identified by Gram staining, urease, catalase and oxidase tests.
Detection of antibody titres
ELISA based Immunocomb II kit for H. pylori IgG (Orgenics, Israel) was used to detect antibodies in 75 serum samples. Manufacturer's instructions were followed and a titre equal to or above the cut off value of 20 u/mL was considered significant. Titres below the cut off value were considered insignificant.
| ~ Results|| |
Out of 75 cases of dyspepsia, rapid urease test was positive in 54 (72%), culture in 21 (28%) and direct microscopy of Gram stained antral biopsy in 15 (20%) cases. Significant IgG titres (> 20u/mL) were detected in 57 (76%) serum samples (Table). All cases which were positive by rapid urease test, had significant IgG levels. Three cases were negative by rapid urease test but had significant IgG levels. No case was detected in which RUT was positive but serology was insignificant.
| ~ Discussion|| |
In the present study, H.pylori could be isolated from 28% cases while smear was positive only in 20% cases. Similar findings have been reported by many workers in India, though some have reported higher results., This low isolation rate may be due to its patchy distribution in gastric mucosa, fastidious nature, mucosal atrophy, intestinal metaplasia (in stomach), administration of antibiotics (due to some other infection or protozoal infestation)4 and proton pump inhibitors. The results of biopsy based rapid urease test in our study were comparable with earlier studies.
The difficulties associated with invasive tests and low isolation rate of this organism has led to the development of various serological tests., Major antigenic determinants have been located on urease enzyme as well as on 128 kDa external protein (Cag A) and cytotoxin (Vac A) of pathogenic H.pylori strains. The ELISA test used in this study is based on the inactivated 128 kDa external protein (Cag A).
Significant IgG titres were detected in 76% cases in this study which is consistent with reports of other authors. Results of positive serology were also comparable with biopsy based rapid urease test which at present is most commonly used test for diagnosis of H.pylori infection. In the present study, three cases were negative by rapid urease test, but had significant IgG levels. This may be due to past infection or patchy distribution of organism in the stomach which was responsible for negative rapid urease test. No case was detected in which rapid urease test was positive but serology insignificant.
Our finding can be explained by the fact that serology assays the systemic response to entire stomach, therefore serological tests may be better in already established (chronic) infections where the organism may not be detected bacteriologically. Therefore, we conclude that H.pylori seropositivity is high among dyspeptic patients in and around Amritsar (Punjab).
However, further studies may be needed in larger number of patients to establish this test as a conclusive procedure. We recommend serology as a screening procedure only and if the titres are insignificant, the role of endoscopy just to make a diagnosis of H.pylori infection in dyspeptic patients is not warranted.
| ~ Acknowledgement|| |
The authors thank Dr. Harpreet Singh, DM (Gastroenterology) for performing gastric biopsies of all 75 patients.
| ~ References|| |
|1.||Warren JR, Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983;1:1273-1275. |
|2.||Calam J. Clinician's guide to Helicobacter pylori: Ist ed. (Chapman & Hall, London) 1996:72-78. |
|3.||Barthal JS, Everett ED. Diagnosis of Campylobacter pylori infections: The gold standard and the alteration. Rev Inf Dis 1990;12:S107 |
|4.||Prasad KN, Singhal S, Ayyagari A, Sharma S, Dhole TN. Association of Helicobacter pylori in patients of Non ulcer dyspepsia. Indian J Microbiol 1991;31:415-418. |
|5.||Malik A, Singhal S, Mukharji U. Evaluation of ELISA based antibody test against H. pylori in cases of non ulcer dyspepsia. Indian J Med Microbiol 1999;17:137-139. |
|6.||Nanivadekar SA, Sawant DD, Saraswathi K, Shroff CP, Bichile LS, Patel HD, Shroff DS. Association of Campylobacter pylori with gastritis, duodenal ulcer and gastric ulcer: a preliminary report of dyspeptic patients. Indian J Gastroenterol 1988;7:141-142. |
|7.||Karnes WE, Samloff IM, Sivrala M. Positive serum antibody and negative tissue staining for H.pylori in subjects with atrophic body gastritis. Gastroenterology 1991;101:167-174. |
|8.||Iwahi T, Satoh H, Nakao M. Lansprazole, a novel benzimidazole proton pump inhibitor, and its related compounds have selected activity against H.pylori. Antimicrob Agents Chemother 1991;35:490-496. |
|9.||Dent JC, McNulty CA, Uffgs, Gear MWL, Wilkinson SP. Campylobacter pylori urease a new serological test. Lancet 1988;1:1002. |
|10.||Covacci, Censini S, Bygnoli M. Molecular characterization of 128 kDa, an immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc Natl Ac Sc, USA 1993;90:5791-5795. |
|11.||Sharma S, Dhole TN, Prasad KN, Ayyagari A. Evaluation of 66 kDa directed IgG response for detection of Helicobacter pylori infection. Indian J Med Microbiol 1996;14:17-21. |