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Table of Contents   
ORIGINAL RESEARCH  
Year : 2012  |  Volume : 23  |  Issue : 5  |  Page : 694-695
Salivary IgG assay to detect Helicobacter pylori infection in an Indian adult population


1 Department of Oral Medicine and Radiology, KSR Institute of Dental Science and Research, Tirchengode, Tamil Nadu, India
2 Department of Oral Medicine and Radiology, Dayananda Sagar College of Dental Sciences and Research, Bangalore, Karnataka, India

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Date of Submission23-Mar-2012
Date of Decision12-Jul-2012
Date of Acceptance11-Oct-2012
Date of Web Publication19-Feb-2013
 

   Abstract 

Background: Helicobacter pylori infection, the commonest chronic bacterial infection in humans, causes chronic gastritis, peptic ulcer, and possibly gastric carcinoma and lymphoma. Recently, investigators have focused on its role in the development of extra-gastrointestinal diseases with oral manifestations. H. pylori infection can be diagnosed by various methods. Of late, H. pylori IgG antibodies have been detected in saliva using enzyme-linked immunosorbent assay (ELISA). However, local validation of serological test is needed before implementing a test in different populations.
Aims: To detect anti H. pylori specific immunoglobulin G (IgG) antibodies in saliva of adult patients with gastrointestinal symptoms by ELISA, to diagnose H. pylori infection in such patients by histopathology, and to evaluate the diagnostic accuracy of the immunoassay as compared to histopathologic diagnosis.
Methods: The study included 40 adult patients with gastrointestinal symptoms suggestive of peptic ulcer disease. Saliva samples were analyzed for anti H. pylori IgG using EIAgen H. pylori IgG kit. Histopathologic diagnosis using gastric biopsy samples was the gold standard.
Results: The sensitivity and specificity of the test were 79.31% and 63.64%, respectively. The positive and negative predictive values were 85.19% and 53.85%, respectively. The accuracy of EIAgen H. pylori IgG kit for salivary detection of anti H. pylori IgG antibodies was found to be 75%.
Conclusion: EIAgen H. pylori IgG assay is a noninvasive, moderately accurate, and sensitive method for the detection of H. pylori infection in saliva. Salivary anti H. pylori IgG test prior to endoscopy is a useful screening test for seroepidemiological studies.

Keywords: endoscopy, Helicobacter pylori, histopathology, IgG, saliva

How to cite this article:
Krishnaswamy RT, David CM, Govindaiah S, Krishnaprasad RB, Jogigowda SC. Salivary IgG assay to detect Helicobacter pylori infection in an Indian adult population. Indian J Dent Res 2012;23:694-5

How to cite this URL:
Krishnaswamy RT, David CM, Govindaiah S, Krishnaprasad RB, Jogigowda SC. Salivary IgG assay to detect Helicobacter pylori infection in an Indian adult population. Indian J Dent Res [serial online] 2012 [cited 2019 Nov 13];23:694-5. Available from: http://www.ijdr.in/text.asp?2012/23/5/694/107416
Helicobacter pylori infection, the commonest chronic bacterial infection in humans, is the major cause of chronic gastritis, peptic ulcer, and possibly gastric carcinoma and lymphoma. [1] Serum immunoglobulin response to the infection is an important determinant of gastric mucosal damage [2] and remains high for months or years unless the infection is treated. Even after eradication of the bacteria, their levels decrease but immunoglobulin G (IgG) does not disappear. Salivary IgG is secreted during the serum immune response, and the levels parallel that of circulatory IgG levels. [3]

Though the definitive diagnosis of H. pylori infection has been based primarily on the isolation of the bacterium in culture or its detection in histological sections of gastric biopsy specimens obtained at endoscopy, [4] they are invasive and expensive. Studies [3],[5],[6],[7] have detected salivary H. pylori IgG antibodies by enzyme-linked immunosorbent assay (ELISA) and have indicated that such a test is moderately accurate. Saliva offers advantages over serum since its collection is easy, noninvasive, and less hazardous, and there is a greatly reduced risk of blood-borne infections. [4] In children, tests based on the detection of antibodies (IgG, IgA) against H. pylori in serum, whole blood, urine, and saliva are not reliable for use in the clinical setting because the sensitivity and specificity for detection of antibodies in them vary widely. [8] In this study on Indian population, we have evaluated the accuracy of EIAgen H. pylori IgG ELISA kit for determination of anti H. pylori IgG in saliva in adults in comparison with histopathology of gastric biopsy samples.


   Materials and Methods Top


The study group comprised 40 adult patients above 18 years of age with gastrointestinal symptoms suggestive of peptic ulcer disease, referred for endoscopy. Patients taking nonsteroidal anti-inflammatory drugs (NSAIDs), bismuth containing drugs, antibiotics, proton pump inhibitors, and corticosteroids at the time of endoscopy or 2 weeks before, and pregnant women were excluded from the study. A brief case history was recorded in a formatted case sheet from the participants, and 40 of them who fulfilled the criteria of selection were finally selected and written informed consent was obtained. About 2-3 ml of unstimulated whole saliva was collected before endoscopy, by aspiration from the floor of mouth or by the passive drool technique. The samples were subjected to centrifugation and the saliva supernatant obtained was stored at −70°C, till the ELISA test was done. Five small bits of gastric biopsy samples obtained by the gastroenterologist from the gastric antrum during endoscopy were immediately fixed using 10% formalin, processed, stained with Giemsa, and histopathologic diagnoses were made. According to the manufacturer, serum samples had to be diluted in 1:101 ratio. But in our study, the saliva samples were diluted in the ratio 1:4 and subjected to ELISA for detection of IgG antibodies to H. pylori using EIAgen H. pylori IgG kit (ADALTIS, Rome, Italy S.p.A). This modification is in accordance with the study by Francesco Luzza et al.[1] The validation of the assay was tested by an ELISA run of the undiluted calibrators supplied by the manufacturer. According to the manufacturer, the cut-off for adult sera should correspond to calibrator 3. As the kit used in this study was a serum kit, but saliva samples were used, the Optical Density (OD) cut-off was calculated by adding two Standard Deviations (SDs) to the mean OD values obtained from saliva samples of 20 adults who were negative for gastrointestinal symptoms and who were subjected to the ELISA procedure. [1]

The value of the OD of the blank was subtracted from the readings of the calibrators to get the net OD value. The OD of calibrator 3 must be ≥0.2 and that of calibrator 5 must be at least 1.5 times that of calibrator 3. The OD of the blank must be ≤0.150. A calibration curve was constructed by plotting in a graph paper, the net OD of each calibrator against its corresponding AU/ml concentration. A standard curve [Table 1], [Figure 1] was obtained and all the values were evaluated according to the manufacturer's instructions. The test results were subjected to statistical analysis (sensitivity, specificity, Chi-square test, t-test, and probability test) using SPSS and Medcalc software (correlation-comparative study).
Figure 1: Standard curve

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Table 1: Net OD values of calibrators

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   Results Top


Esophagitis, antral gastritis, bulbar duodenitis, gastric and duodenal ulcers were diagnosed endoscopically in 38 out of 40 patients. Two patients had normal mucosa endoscopically. H. pylori infection was confirmed by histopathologic diagnosis in 29 out of 40 patients.

Results of salivary ELISA were evaluated according to the manufacturer's instructions. Twenty-seven out of 40 patients were positive for the presence of anti H. pylori antibodies in the saliva and 13 were negative. But out of 29 H. pylori positive cases confirmed by histopathology, 23 were positive for the presence of anti H. pylori antibodies in the saliva and 7 out of 11 H. pylori negative cases were negative for the presence of anti H. pylori antibodies in the saliva. There were four false-positive and six false-negative results [Table 2], [Figure 2]. Based on these results, the sensitivity of the test was found to be 79.31% and specificity was 63.64%. The positive and negative predictive values (PPV and NPV) of the test were found to be 85.19% and 53.85%, respectively.
Figure 2: Comparison of ELISA and histopathology results

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Table 2: Comparison of ELISA test results with histopathology result

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Statistically significant association was observed between ELISA test results and histopathology results (P < 0.05) [Table 3]. Higher mean OD was recorded in H. pylori positive patients compared to H. pylori negative patients [Table 4], but the difference in mean OD was not statistically significant (P > 0.05) [Figure 3]. The accuracy of the EIAgen H. pylori IgG kit for salivary detection of anti H. pylori IgG antibodies was found to be 75%.
Figure 3: Mean OD values in positive and negative samples

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Table 3: Association between ELISA test results and histopathology results

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Table 4: Comparison of OD (AU/ml) between positive and negative H. pylori patients (based on results from histopathology)

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   Discussion Top


Screening for and detection of H. pylori infections are of great significance due to a huge discrepancy in their prevalence rates and incidence. H. pylori infection varies widely between the industrialized countries and the rest of the world. All the studies which focused on evaluating the tests used to detect H. pylori faced the problem of an appropriate gold standard. However, to evaluate the validity of a noninvasive test, one would consider invasive tests as the reference. [4] In our study, the histopathologic diagnosis was taken as the gold standard because it is the most widely available method, and it allows an evaluation of the status of gastric mucosa such as the presence of atrophy, intestinal metaplasia, lymphoid follicles, dysplasia, and carcinoma. It is relatively easier and a reliable method compared to the other invasive methods used in previous studies. [1],[3],[5],[6],[7],[9],[10]

The objectives of our study were to detect anti H. pylori specific IgG antibodies in the saliva of adult patients with gastrointestinal symptoms by ELISA, to diagnose H. pylori infection in such patients by histopathology, and to evaluate the diagnostic accuracy of the immunoassay as compared to histopathologic diagnosis. Twenty-nine out of 40 study subjects in our study tested positive for H. pylori in histopathology, as against a study in which [2] H. pylori was detected in only 31 patients out of 97. This can be explained by the fact that H. pylori can be visualized at high magnification with conventional hematoxylin and eosin (H and E)-stained sections, but H and E staining may be unreliable when few bacteria are present. In addition, luminal debris on the surface of the epithelium can be mistaken for H. pylori in H and E-stained sections. Histological identification of bacteria is facilitated by using special stains such as the Warthin-Starry and modified Giemsa stains. [11] As the routine H and E stain is not well suited for H. pylori detection, we used Giemsa stain which yielded better results in histopathologic diagnosis compared to the study that used Gram stain.

In our study, anti H. pylori antibodies in saliva were detected in 27 patients by ELISA (67.5%). In a study, [3] 60 saliva samples were positive out of 97, i.e. 62%. In another study, [9] Helisal salivary immunoassay was used and saliva anti H. pylori antibodies were detected in 104 subjects out of 195, i.e. 52.7%. Compared to these studies, our study has shown slightly higher sensitivity. As it is important to store the saliva at proper temperature to prevent salivary antibody degradation, in our study we stored the samples at −70°C, which may be one of the reasons for this result.

The salivary dilution factor significantly influences the assay results. A study on adults [1] compared the salivary IgG assay to histopathology diagnosis and used the working dilution of 1:4; the sensitivity and specificity of salivary H. pylori IgG were 82% and 71%, respectively. The PPV and NPV were 95% and 40%, respectively, and the accuracy was 81%. Owing to the common reference standard, and the higher PPV and accuracy, with a dilution of 1:4, we have adopted the same dilution factor of 1:4.

The results of our study are consistent with those of the studies on detection of H. pylori IgG in saliva of adult patients using commercial enzyme immune assays developed for detection of serum H. pylori antibodies in the past, which have reported sensitivities and specificities in the range of 66-85% and 58-74%, respectively, compared to histopathologic diagnosis. [4] Our results are also consistent with the study [9] where Helisal assay was used with accuracy of 78% and sensitivity of 81%, compared to culture and/or histology methods. But the specificity of their study was much higher, i.e. 75%. They used specific enzyme immunoassay for saliva samples and/or the use of Omni-SAL saliva collection device in their study as such specialized collection devices enhance the level of antibodies, particularly IgG, in oral specimens, ensure sufficient specimen volume, and include reagents to prevent microbial growth and proteolytic breakdown of antibodies. [12] As Salimetrics [13] recommends that unstimulated whole saliva collected by the passive drool technique is the best type of saliva to use for most general studies, we used the same instead of the specialized collecting devices which collect specific glandular saliva. The higher PPV of our study (85.19%) compared to 76% in their study [9] is probably because they stored the salivary samples at room temperature at which salivary antibody degradation is more likely.

In our study, the overall sensitivity and specificity were 79.3% and 63.64%, respectively, whereas those of gastritis and peptic ulcer (GAP) test ELISA, [5] against the histological, culture, and biochemical detection of the bacterium, were 84% and 90%, respectively. One probable reason for these better results in GAP test ELISA could be the methodology of salivary ELISA. Also, the British Society of Gastroenterologists [14] has compared the sensitivity, specificity, and predictive values of six commercial serological kits including GAP test Bio Rad which were intended for pre-endoscopy screening of H. pylori infection and which were compared with histopathology and culture tests. Accordingly, it was reported that this test performed with excellent sensitivity and NPV. This aspect of the kit could have yielded higher specificity of 90% in their study.

A recent study [15] has compared the performance of two indirect ELISAs detecting H. pylori specific IgG antibodies in serum and saliva with endoscopic observations and histological findings of biopsies from dyspeptic patients, using specific enzyme immunoassay for saliva and serum samples (Helisal TM saliva and Helisal TM serum) and Omni-SAL saliva collection device. In addition, prior to testing, saliva specimens from all patients were vortex-mixed for 1 min, the pads were further squeezed, and the fluid was carefully pipetted from the bottom of the tubes to avoid air bubbles. The sensitivities of the serum and saliva tests were 90.5% and 95%, respectively, while the specificities were 84.5% and 70%, respectively. As we did not adhere to such specialized devices and techniques, our study has shown lesser sensitivity and specificity.

The variation in sensitivities and specificities in different studies can be explained based on the standard H. pylori detection test taken for comparison [as illustrated in [Table 5]]. [16] This could contribute to the variable results of salivary immunoassays for which they have been compared.
Table 5: Variation in sensitivities and specificities of different H. pylori detection tests

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The variability of results between kits may also be due to the different antigen preparations. Further, the commercially available ELISA kits are not uniformly effective in different geographic regions and their criteria are lower in East Asia than in western countries. [17] This could be one of the reasons for the better results of salivary assay performed using the laboratory antigens [3] and the in-house ELISA [1] on Iranian and Italian populations, respectively. As the EIAgen H. pylori IgG kit used in our study is an Italian kit, the validity of the assay when used on Indian population might vary. The choice of cut-off value affects the assay's specificity and sensitivity in a reciprocal manner, with a major impact on the clinical decision-making process. [5] The cut-off of 0.2 obtained in our study is comparable to that used in other studies. [1],[5]

Several factors might have contributed to the false-positive and false-negative results. Some authors have emphasized the need to use local strains as the source of antigen rather than foreign strains to obtain the best test performance. [3] The false-positive results can be due to unknown cross-reacting bacterial antibodies. Though the analytical specificity of the kit used in our study has been evaluated in an inhibition test and has been concluded that the test is specific for anti H. pylori IgG in serum, the specificity of the test kit to saliva H. pylori antibodies has not yet been established. Though our study has excluded patients on omeprazole at the time of endoscopy or 2 weeks before, the fact that omeprazole administered as a monotherapy for 4 weeks decreases the bacterial density in both the antrum and the corpus, but at differing magnitudes from one patient to another may support the probability. Antibiotics used as monotherapy also have a dramatic effect on the bacterial density even if they rarely lead to H. pylori eradication. The very slow decrease in antibodies after eradication (25% in titer within 6 months or more) may also be a cause of a false-positive result. [4] False-negative results may occur following a new infection before the antibody level is sufficiently elevated. In developing countries, the specificity of the test may be altered by the presence of concomitant infections, especially with campylobacter or other related bacteria; furthermore, the host immune response may be lower due to malnutrition. [18] In our study, one case had history of tuberculosis, and owing to the immunosuppressive state, the antibody response might have been lowest, thus causing a false-negative result. However, out of 23 true positives in our study, saliva ELISA could detect anti H. pylori antibodies in 9 cases diagnosed as bulbar duodenitis, 10 as antral gastritis, and 3 as duodenal ulcers endoscopically. Although one case was endoscopically a normal mucosal study, histopathology was positive for H. pylori infection and saliva H. pylori antibodies were also detected by ELISA.

Several factors need to be considered for a reasonably accurate salivary detection of H. pylori IgG antibodies, such as the type of kit to be used, source of antigens, and the complexity of assay protocols. A combination of different antigens, especially high molecular weight, cell-associated proteins, including the Cytotoxin associated Antigen (CagA) protein (120 kDa) appears to be superior in terms of sensitivity and specificity. The most important is collection of the saliva samples and their proper handling as contaminated samples may affect the assay result. The dilution factor for the salivary samples has not yet been standardized for salivary detection of H. pylori antibodies by ELISA, and also the development of specific enzyme immunoassays for salivary detection of such antibodies is still in the embryonic stage.


   Conclusion Top


It has been suggested that serological tests for H. pylori should be locally validated because assays validated in one region may yield variable diagnostic performances in others. [19] Therefore, reevaluation is needed before implementing a test in different populations. [20] EIAgen H. pylori IgG assay used in our study on Indian population is moderately accurate for the detection of H. pylori antibodies in symptomatic individuals. Our results show that the salivary ELISA is a sensitive method, and taking into account the advantages of saliva over serum, it can be considered as a diagnostic aid provided the other factors determining the accuracy of the test are addressed.

 
   References Top

1.Luzza F, Oderda G, Maletta M, Imeneo M, Mesuraca L, Chioboli E, et al. Salivary immunoglobulin G assay to diagnose Helicobacter pylori infection in children. J Clin Microbiol 1997;35:3358-60.  Back to cited text no. 1
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2.Gasbarrini A, Franceschi F, Armuzzi A, Ojetti V, Candelli M, Torre ES, et al. Extradigestive manifestations of Helicobacter pylori gastric infection. Gut 1999;45:i9-i12.  Back to cited text no. 2
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3.Ghasemian Safaei H, Fazeli A, Tamizifar H, Rashidi N. Comparison of salivary anti Helicobacter pylori IgG with serum IgG and bacteriological tests in detecting Helicobacter pylori infections. J Res Med Sci 2005;1:1-5.  Back to cited text no. 3
    
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5.De Pascalis R, Del Pezzo M, Nardone G, Budillon C, Lavitol A. Performance characteristics of an enzyme-linked immunosorbentassay for determining salivary immunoglobulin Gresponse to Helicobacter pylori. J Clin Microbiol 1999;37:430-2.  Back to cited text no. 5
    
6.Hooshmand B, Monsef A, Amirmadglesi M, Kashani M. Oral fluid antibody detection in the diagnosis of gastric Helicobacter pylori infection. Iran J Allergy Asthma Immunol 2004;3:127-31.  Back to cited text no. 6
    
7.Bode G, Marchildon P, Peacock J, Brenner H, Rothenbacher D. Diagnosis of Helicobacter pylori Infection in children: Comparison of a salivary immunoglobulin G antibody test with the [13C] urea breath test. Clin Diagn Lab Immunol 2002;9:493-5.  Back to cited text no. 7
    
8.Koletzko S, Jones NL, Goodman KJ, Gold B, Rowland M, Cadranel S, et al. Evidence-based Guidelines From ESPGHAN and NASPGHAN for Helicobacter pylori Infection in Children. J Pediatr Gastroenterol Nutr 2011;53:230-43.  Back to cited text no. 8
    
9.Simor AE, Lin E, Saibil F, Cohen L, Louie M, Pearen S, et al. Evaluation of enzyme immunoassay for detection of salivary antibody to Helicobacter pylori. J Clin Microbiol 1996;34:550-3.  Back to cited text no. 9
    
10.Patel P, Mendall MA, Khulusi S, Molineaux N, Levy J, Maxwell JD, et al. Salivary antibodies to Helicobacter pylori: Screening dyspeptic patients before endoscopy. Lancet 1994;344;511-2.  Back to cited text no. 10
    
11.Dunn BE, Cohen H, Blaser MJ. Helicobacter pylori. Clin Microbiol Rev 1997;10:720-41.  Back to cited text no. 11
    
12.Hodinka RL, Nagashunmugam T, Malamud D. Detection of human immunodeficiency virus antibodies in oral fluids. Clin Diagn Lab Immunol 1998;5:419-26.  Back to cited text no. 12
    
13.Advice on Measuring SIgA in Saliva.101 Innovation Boulevard, Suite 302, State College, PA 16803, USA: Salimetrics 2010.  Back to cited text no. 13
    
14.McNulty CA, Nair P, Watson BE, Uff JS, Valori RM. A comparison of six commercial kits for Helicobacter pylori detection. Commun Dis Public Health 1999;2:59-63.  Back to cited text no. 14
    
15.El-Mekki A, Kumar A, AlknawyB, Al-Ammari O, Moosa R, Quli S, et al. Comparison of enzyme immunoassays detecting Helicobacter pylori specific IgG in serum and saliva withendoscopic and biopsy findings in patients with dyspepsia. Indian J Med Microbiol 2011;29:136-40.  Back to cited text no. 15
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16.Monteiro L, de Mascarel A, Sarrasqueta AM, Bergey B, Barberis C, Talby P, et al. Diagnosis of Helicobacter pylori infection: Noninvasive methods compared to invasive methods and evaluation of two new tests. Am J Gastroenterol 2001;96:353-8.  Back to cited text no. 16
    
17.Mohammedi M, Talebkhan Y, Khalili G, Mahboudi F, Massarrat S, Zamaninia L, et al. Advantage of using a home made ELISA kit for detection of Helicobacter pylori infection over commercially imported kits. Indian J Med Microbiol 2008;26:127-31.  Back to cited text no. 17
    
18.Baysoy G, Ertem D, Ademoglu E, Kotiloðlu E, Keskin S, Pehlivanoðlu E. Gastric Histopathology, iron status and iron deficiency anemia in children with Helicobacter pylori infection. J Pediatr Gastroenterol Nutr 2004;38:146-5.  Back to cited text no. 18
    
19.Deankanob W, Chomvarin C, Hahnvajanawong C, Intapan PM, Wongwajana S, Mairiang P, et al. Enzyme-linked Immunosorbent Assay for Serodiagnosis of Helicobacter pylori in dyspeptic patients and volunteer blood donors. Southeast Asian J Trop Med Public Health 2006;37:858-965.  Back to cited text no. 19
    
20.Marchildon PA, Ciota LM, Zamaniyan FZ, Peacock JS, Graham DY. Evaluation of three commercial enzyme immunoassays compared with the 13C urea breath test for detection of Helicobacter pylori infection. J Clin Microbiol 1996;34:1147-52.  Back to cited text no. 20
    

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Correspondence Address:
Ramya Thirumala Krishnaswamy
Department of Oral Medicine and Radiology, KSR Institute of Dental Science and Research, Tirchengode, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.107416

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