Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 2, 2011, pp. 136-140

Indian Journal of Medical Microbiology, Vol. 29, No. 2, April-June, 2011, pp. 136-140

Original Article

Comparison of enzyme immunoassays detecting Helicobacter pylori specific IgG in serum and saliva with endoscopic and biopsy findings in patients with dyspepsia

A El-Mekki¹, A Kumar¹, B Alknawy², O Al-Ammari³, R Moosa¹, S Quli¹, M Ahmed⁴

¹ Department of Clinical Microbiology and Parasitology, King Khalid University, College of Medicine, Abha, P.O. Box 641, Pin - 61421, Kingdom of Saudi Arabia
² National Guard Health Affairs, Riyadh, Kingdom of Saudi Arabia
³ King Faisal Specialist Hospital and Research Center, Jeddah, Kingdom of Saudi Arabia
⁴ Faculty of Medicine, University of Medical Sciences and Technology, P.O. Box.12810, Khartoum, Sudan

Correspondence Address: A Kumar Department of Clinical Microbiology and Parasitology, King Khalid University, College of Medicine, Abha, P.O. Box 641, Pin - 61421 Kingdom of Saudi Arabia vashish2010@gmail.com

Date of Submission: 06-Dec-2010
Date of Acceptance: 30-Jan-2011

Code Number: mb11032

PMID: 21654107

DOI: 10.4103/0255-0857.81793

Abstract

Keywords: Antibodies, dyspepsia, H. pylori, serum, saliva

Introduction

Infection with Helicobacter pylori (HP) is particularly common in developing countries of Asia with prevalence rates approaching 95% in patients with dyspepsia. ^[1]

Recent studies of a number of serologic tests currently available for diagnosing HP infection seem to indicate that none of them is ideal and therefore, the performance characteristics of any of these tests should be carefully considered prior to their utilization in a given setting. ^[2],[3] For quick office evaluation of dyspepsia and population-based studies, a simple, rapid, reliable and an inexpensive test is highly desirable. ^[4] Because of their reported high sensitivity and specificity, ^[5],[6] commercially available antibody tests seem to be well suited for epidemiological surveys, practically among children and untreated patients. ^{[7],[8],[9],[10]} Since the pattern of HP infection in the developing world is quite distinct from that in the Western countries ^[11] and because the economic constraints make invasive tests difficult to perform, the search for reliable and cheap tests that suit such situations becomes necessary. Thus our aim in the present study was to compare and evaluate the findings of two indirect enzyme immunoassays detecting HP-specific IgG class antibody levels in saliva and serum, with endoscopic observations and histologic biopsy findings as a standard test, hoping to select a suitable non-invasive test capable of accurately diagnosing HP infection in our community.

Materials and Methods

Patients and clinical specimens

Fifty five patients (20 males, 35 females; mean age 37 years) with dyspeptic symptoms, consecutively attending the endoscopy unit of the hospital were enrolled in the present study. None of these patients had been on antibiotics previously. About 10 ml of venous blood was collected in plain tubes from each patient. Saliva was also collected from patients by placing the absorbent pad in the mouth using Omni-SAL ^TM Saliva Collection Device until the indicator turned blue (Omni-SAL ^TM Saliva Diagnostic Systems Ltd., Dartford, England, UK). These samples were kept in the collection tubes and stored at 4° C. Separated sera were aliquoted and kept frozen until tested.

Endoscopic diagnosis was initially recorded from each patient and subsequently, an average of three mucosal biopsies were taken from within 5 cm of the pylorus. Biopsies were kept in 10% buffered formalin until it was further processed for histopathologic examination and detection of HP.

Methods

Two indirect quantitative enzyme immunoassays (HeliSAL ^TM saliva and HeliSAL ^TM serum, Cortecs Diagnostics Ltd., Techbase 1, Newtech Square, Deeside Industrial Park, UK CH5 2NT, Clwyd, UK) were utilized for the detection of HP-specific IgG class antibodies in saliva and serum, respectively. Prior to testing, saliva specimens from all patients were vortex-mixed for one minute, the pads were further squeezed and the fluid was carefully pipetted from the bottom of the tubes to avoid air bubbles. The specimens were then transferred onto antigen-coated plates. Frozen serum samples from all patients were allowed to thaw at room temperature prior to testing. Enzyme immunoassays were then performed and evaluated according to the manufacturer′s instructions. In all runs, standard curves were plotted and the IgG concentration in tested samples, expressed as units, were directly read against their optical density. Values ≥1 units/ml were considered positive for HP-specific IgG antibodies in serum and saliva, respectively. Biopsies from patients were stained with hematoxylin and eosin and Giemsa stain and examined for histopathological changes and presence or absence of HP. Laboratory data thus collected from patients were analyzed by Prism ^TM version 2.0 (GraphPad Software, Inc, San Diego, USA).

Results

Of the biopsies obtained from the 55 patients with dyspeptic symptoms, 42 (76.36%) were positive for HP on staining. These microorganisms were identified on stained sections from biopsies in 4 of the 16 (25%) cases with normal endoscopic findings, in all 16 cases of gastritis and in 22 of the 23 (95.6%) cases with duodenal ulcers [Table - 1]. Prevalence of HP was significantly higher in biopsies from patients with gastritis (P = 0.0001) and duodenal ulcers (P = 0.0001) compared to those with normal endoscopic findings. Serum HP-specific IgG antibodies were detected in all the 4 cases with normal endoscopy findings with positive biopsies, in 12 of the 16 cases of gastritis and in 22 of the 23 cases of duodenal ulcers with positive biopsies. There were two false positives among those with normal endoscopic findings, and four false negatives among cases with gastritis. In saliva, specific IgG antibodies were detected in all the 4 cases with positive biopsies and in 4 additional cases with negative biopsies (i.e. false positives), in 14 of the 16 cases of gastritis and in all biopsy-positive cases with duodenal ulcers. Prevalence of specific antibodies in either sera or saliva did not show significant differences between normals and those with gastritis (P = 0.0730, 0.0538, respectively). However, a significant higher prevalence of these antibodies was observed in those with duodenal ulcers when compared to normals (P = 0.0001 and 0.0015 for serum and saliva, respectively). The sensitivity and specificity of HeliSAL ^TM serum and HeliSAL ^TM saliva were 100% in the latter group. The overall performance characteristics of these immunoassays are summarized in [Table - 2].

Column scatters plotted in [Figure - 1] and [Figure - 2] represent serum and saliva IgG concentrations, respectively. Statistical analysis utilizing t tests and two-tailed P values did not show any significant difference (at 95% CI) between mean IgG concentrations [Figure - 3] of normals compared to those with gastritis or duodenal ulcers.

Discussion

Many endoscopy units, including ours, adopt an open access policy and therefore receive requests for upper gastrointestinal endoscopy (UGE) at a rate that exceeds the available facilities, ^[12] hence the availability of a simple and a reliable test will significantly reduce the load of UGE by screening the large number of young patients presenting with dyspepsia. ^[13],[14] Thus serologic tests of serum or saliva seem to be an attractive alternative that will reduce UGE load as well as allow the empirical treatment of patients with antisecretory and antimicrobial agents, thereby greatly reducing the cost of patient management. ^[15]

The present study compared the performance of serum and saliva HP-specific IgG serology to antral biopsy histopathology, which is considered one of the gold standard methods for HP detection in a population with high prevalence of HP infection. ^[1],[16] The performance of both serum and saliva immunoassays were concordant with the detection of HP in biopsies from patients with DU and approaches the findings in patients with gastritis. These results are similar to previous reports, which also documented the good sensitivity and specificity of these assays, ^[6],[17] but a recent report from Canada demonstrated a low sensitivity and specificity of the saliva ELISA test. ^[18] However, these differences can be attributed to several factors such as the type of population studied, decreased saliva flow due to pre-endoscopy anxiety, or differences of HP strains. Thus, if endoscopy had not been done, none of the HP-associated DU and very few of the gastritis group would have been missed by either serologic tests. In this respect, our findings were better than those reported by Reilly and co-workers who found that about 10 to 15% of HP-related pathology could be missed by those tests. ^[5] The false-positive results observed in some patients with normal endoscopy findings could be explained by the nature of our population, being patients referred with long standing dyspepsia and are therefore expected to have high prevalence of HP (76.4%), which could have decreased the negative predictive value of these tests. ^[19] This trend could also have arisen from possible strain cross-reactivities, sampling errors, differences in defining the cut-off values or denial of some patients with normal endoscopy or recent consumption of drugs. ^{[1],[19],[20]} Interestingly, we found no significant difference in the quantity of antibody in either serum or saliva between the normals and those with gastritis or duodenal ulcers [Figure - 3] but a significant difference in the prevalence of these antibodies was observed between the normal group and patients with DU (P = 0.0001 and 0.0015 for serum and saliva, respectively). This supports the usefulness of these tests in detecting HP-associated DU patients and contrasts previous findings which suggested that serum IgG assays were much better than saliva IgG in detecting such patients. ^[21] The performance characteristics of the enzyme immunoassays currently used in detecting salivary or serum IgG can be further refined by introducing IgG capture immunoassays utilizing highly specific enzyme-labeled HP antigens.

In conclusion, in spite of the limitations of this study, we are of the opinion that immunoassays detecting HP-specific IgG antibodies in saliva are simple and practical in clinical settings and are concordant with invasive tests. ^[9] Furthermore, as suggested by some authors, such tests, can be considered as good as the gold standard biopsy test in diagnosing HP infection and screening of dyspeptic patients. ^[22],[23] In settings similar to ours, we would therefore recommend the use of HP-specific salivary IgG assays, which offer a simple means of sample collection, especially in children and in difficult patients who usually resent venipuncture for blood sampling, although the issue of how best to diagnose HP infection remains a matter of concern ^[24] as do the many gray areas in the microbiological and clinical aspects of HP infection. ^[25]

References

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