Non-Invasive Diagnosis of Helicobacter pylori: Evaluation of Two Enzyme Immunoassays, Testing Serum IgG and IgA Response in the Anand District of Central Gujarat,India

Context: Validation of an accurate and less cumbersome noninvasive method to detect current Helicobacter pylori infection is a requisite for any laboratory.

Objectives: The purpose of this study is to corroborate the usefulness of two commercially available kits NovaTec ELISA-A and ELISA-G, in the detection of ongoing H.pylori infection.

Materials and Methods: Two hundred and twenty eight condivutive serum samples of symptomatic patients who attended the endoscopy unit of “Deep” surgical hospital, Anand, which were collected during the period from 27th February 2008 to 31st august 2011, were studied. The sera were processed and tested for the detection of the H.pylori IgG and IgA antibody by using a solid phase; capture micro well ELISA, procured from Nova Tec immunodiagnostica GmbH Germany.

Results: IgG ELISA showed 100% sensitivity and Negative predictive value (NPV), while IgA ELISA was better in terms of specificity (61.4%) and accuracy (63%) as compared to IgG ELISA. We found 7% (16/228) of IgA positive cases with IgG negative response. IgG response was more common in reflux esophagitis patients (OR 1.451, 95%CI-0.850-2.477) and then in gastritis (OR 0.962, 95%CI-0.570-1.621) and duodenitis(OR-0.806, 95%CI-0.112-5.827), while IgA positive response was more common in duodenitis patients (OR-1.383, 95%CI-0.191-9.995) and reflux esophagitis patients (OR 1.289, 95% CI-0.756-2.197) and least in duodenal ulcer patients (OR 0.670, 95%CI-0.222-2.029).

Conclusion: IgG update is reliable and accurate test and can be expedient as a screening test and thus serve as an alternative to endoscopy. For the purpose of excluding infection with H.pylori, the performance of IgG is moderate (low specificity) but can be improved by conjunctional IgA testing which will offer some additional diagnostic value.

Helicobacter pylori, in a very short period of time, has become one of the most important etiological agent causing gastrointestinal infections. This organism has dramatically affected gastroenterology and has been a topic of much research. It is now felt to play major roles in the reoccurrence or pathogenesis of duodenal ulcer diseases, chronic antral gastritis, gastric ulcer diseases and, possibly, non-ulcer dyspepsia and gastric cancer [1]. The prominence of H.pylori makes it imperative to develop a safe, noninvasive simple method of detection [1].

The most indicative and accurate method of H.pylori diagnosis is endoscopy followed by colorimetric assays. Most of the laboratories in Central Gujarat confer the results mostly on the basis of IgG serology and only those experiencing intolerable symptoms with indications of progressive disease are referred for endoscopy. This method of H.pylori diagnosis, is quite unappealing and is met by severe hesitations due to its invasive nature. A simple cost-effective diagnostic procedure which would bypass the need for endoscopy is highly required for a population like Central Gujarat in which the rate of infection is overwhelming.

Serologic testing is the commonest method of non-invasive diagnosis for H.pylori used in epidemiological studies, enabling us to screen patients suffering from dyspepsia before performance of endoscopies and for long term follow-ups subsequent to drug therapy Section[1]. When performing serologic testing, however, the physician does need to keep in mind the sensitivity, specificity, and predictive value of the test, parameters that depend on the prevalence of the disease in the population being tested. Generally the prevalence of raised IgG in the population tends to be higher in developing countries than in developed countries [2]. When prevalence of the disease is high, a sensitive test might be chosen, whereas when the prevalence of disease is low, a specific test is more appropriate [2]. Several reports have convincingly demonstrated that changes in H.pylori IgG levels can be used to monitor the success of antibiotic treatment. Nearly all studies have found that successful treatment is associated with a 40-50% decrease in IgG levels by 6 months post-treatment. However, only about 25% of successfully treated patients show a complete disappearance of IgG antibodies even measured 3.5 years after treatment [3].

The clinical importance of the IgA response is underlined when considered in association with earlier findings showing an association between H.pylori IgA and gastric cancer, with increased risk of peptic ulcer disease, atrophic gastritis and intestinal metaplasia [3]. As the H.pylori IgA response may develop later in life, many H.pylori infected young individuals may have IgG only, although this does not exclude the possibility of developing an IgA response [3]. Commercial serological assays for H.pylori detection demonstrate varying accuracies for different populations which may be due to the vast molecular diversity in genes and encoded proteins among isolated H.pylori strains from different geographic regions used for the coating antigen preparations [4].

In Anand District, it has been observed that majority of the individuals are suffering from dyspeptic symptoms attending the gastroenterologist, it is therefore very important to rule out the presence of H.pylori infection. So in the present study the corroboration of both IgG and IgA antibodies in the infected serum are done in comparison to the Gold standard tests, so as to preclude on giving false treatment to the patients.

Subjects

Two hundred twenty eight consecutive, symptomatic patients (144 males and 84 females; age 10-90 years) attending the endoscopy unit of Deep Surgical Hospital, Anand, Gujarat, were included. 115 patients with gastritis, 94 patients with reflux esophagitis, 15 with duodenal ulcer, and 4 patients with duodenitis, based on endoscopic findings were enrolled in this study. Patients taking aspirin or non-steroidal anti-inflammatory drugs (NSAIDS) in the past 4 weeks or those on proton pump inhibitors (PPI), patients with previous therapy to eradicate H.pylori or if the informed consent was not obtained were excluded from the study. The study was conducted in the Department of Microbiology of Shree P. M. Patel Paramedical College, Anand, (India) during the period from February 2008 to August 2011.

Ethical considerations

Approval from Human research ethics committee (HREC) of H. M. Patel center for medical care and education, Pramukh Swami Medical College, Karamsad, was taken prior to initiation of the work. Study was done according to the principles of Helsinki Declaration. Dully filled consent form was obtained from all the patients participating in the study.

Criteria for true positive result for H.pylori: (Gold standard tests)

Subjects were classified as having current infection with H.pylori if Rapid urease test (RUT) and histology were positive along with Gram’s staining or if H.pylori were cultured from the biopsy specimen, if any single test out of RUT, Gram staining or histology was positive then the result was considered negative.

Sampling

Four antral biopsies were collected from each symptomatic patient in Brain-heart infusion (BHI) broth and were transported to the laboratory without delay.

For Gram staining- biopsy was crushed and smears were prepared on a clean slide and stained with the standard protocols [5]. For RUT, one biopsy specimen was immediately placed into 0.5 ml urea indicator broth at room temperature. Any change in color from yellow-orange to pink in the next four hours indicated the presence of H.pylori in the sample [6]. H.pylori were isolated by streaking homogenized biopsies on Brucella blood agar(Brucella Hi Veg Agar Base, procured from Hi-Media labs) augmented with 7-10% human defibrinated blood and Skirrow’s selective supplement (vancomycin, 10μg/ml; polymyxin B sulfate 2.5 IU /ml; trimethoprim lactate 5 μg/ml)(campylobacter supplement-III, Hi-Media labs). These plates were incubated at 37OC in an anaerobic jar with providing gas pack kit (campylobacter gas generating kit BR 060A, Oxoid) for H.pylori which provides suitable microaerophilic condition. H.pylori isolates were identified by typical colony morphology (minute, translucent, round, convex colonies on Brucella blood agar) and characteristic gram negative spiral appearance, positive urease, oxidase and catalase test [7].

Histology slides were stained with Giemsa and Warthin-starry and scored for the presence of H.pylori in several fields [8].

Serology

For serological studies 5mL of venous blood was also obtained from every subject. Serum samples were separated and stored at 4°C until ELISA was done (2 days), after which the samples were stored at 20°C. Specific IgA and IgG antibodies against H.pylori were measured using enzyme linked immunosorbant assay (ELISA). Kits were procured from Nova Tec immunodiagnostica GmbH, Germany.

Antigens used for IgA: recombinant Cytotoxin associated gene (CagA) - and recombinant urease-antigens. Antigens used for IgG: Highly purified proteins associated with CagA genes (120 KD) and Vacuolating (VacA) genes (87 KD) as well as urease-antigens. Procedure of ELISA was according to the manufacturer’s instructions. Results were interpreted as reactive if the absorbance value is >20NTU/ml, Non-Reactive if value is <15 NTU/ml and in Grey zone if value is between 15-20NTU/ml.

Of 228 patients, 18 were H.pylori positive and 210 were H.pylori negative by using the results of the biopsy test as the “Gold standard”. Out of 18 positive samples EIA-G detected all the 18, while EIA-A detected 15 samples positive with 3 false negative samples. One hundred eight samples were false positive with EIA-G update while 81 were false positive with EIA-A update. This result shown in [Table/Fig-1] reveals that the EIA-G update had an optimal sensitivity and negative predictive values of 100% but with reduced specificity of 48.6%. EIA-A update performed better in terms of specificity. EIA-A yielded the most false negative results (46 samples). If we see the combine result comparing to the gold standard tests then, 80 samples were positive by both IgG and IgA while 86 samples were negative by both. Fourty-six samples were positive by IgG but negative by IgA and 16 samples were positive by IgA but negative by IgG [Table/Fig-2] .

Antibody responses in various gastro duodenal diseases [Table/Fig-3] :

IgG response was more common in reflux esophagitis patients (OR 1.451, 95%CI-0.850-2.477) and then in gastritis (OR 0.962, 95%CI-0.570-1.621) and duodenitis (OR-0.806, 95%CI-0.112-5.827) and last in duodenal ulcer (OR 0.271, 95% CI-0.084-0.879).

IgA response was more common in duodenitis patients (OR-1.383, 95%CI-0.191-9.995) and reflux esophagitis patients (OR 1.289, 95% CI-0.756-2.197) and least in duodenal ulcer patients (OR 0.670, 95%CI-0.222-2.029). Even though the Overall IgG and IgA response out of 228 patients was highest in the gastritis patients (63 vs.46), followed by reflux esophagitis patients (57 vs. 43), while it was least in duodenitis and duodenal ulcer.

In view of the patchy distribution of H.pylori, all biopsy-based tests may theoretically fail to diagnose the infection. In contrast to biopsy-based methods, non-invasive tests assess the global presence of H.pylori in the stomach even when the bacteria are irregularly distributed on the gastric mucosa and it also obviates the need for endoscopy [9]. H.pylori infection provokes both local and systemic antibody responses. The systemic response typically comprises a transient rise in IgM, followed by a rise in specific IgA and IgG maintained throughout infection [10]. Almost all H.pylori infected individuals have elevated levels of specific IgG antibodies, but only in about two-third of cases does the IgA titer exceed the cut-off level [1].

The IgG, evaluated in our study, had a sensitivity and negative predictive values of 100% respectively, which is almost in accordance with other studies [11-13] [Table/Fig-4] . The high sensitivity observed permits the safe use of the test in epidemiologic surveys. Although we got very less specificity (108 samples were false positive), indicating that the ELISA must be validated for different populations. As it is proved earlier that sensitivity and specificity of serology depends on the gold standards used to compare the tests, the nature of the antigens employed and the value chosen for the cutoff and it is highly variable, ranging from 30%-100% [11]. Certainly other variables than this might be responsible for the differences observed between various studies, such as the severity and staging of peptic disease and the different strains of microorganism might also play a role [14].

Our study also substantiates that the association between IgG and IgA will result in a marked improvement of the negative predictive value in comparison with the two assays alone but will not offer advantages in relation to positive predictive value .The 100% NPV of IgG makes it useful as a screening test and thus serves as an alternative to endoscopy.

In agreement with earlier reports [15-17] the results of the present study show that there is a significant association between detection of antibody and H.pylori infection. When both the IgG & IgA are negative H.pylori infection is also negative and the proportion of H.pylori infection is higher in patients who had both IgG and IgA positive result(p-value - 0.01, significant).

IgG response was more common in reflux esophagitis patients and then in gastritis [Table/Fig-3] . This data states that IgG response is associated with increase (1.451 times) risk of reflux esophagitis, followed by gastritis and duodenitis. Wyatt et al., [18] also suggested that the serological index of IgG antibodies against H.pylori is related to the severity of antral gastritis and the density of antral colonization.

The results of IgA ELISA are very well correlated with other studies [13,14] [Table/Fig-5] . Although IgA ELISA showed lower sensitivity compare to IgG ELISA [Table/Fig-1] because most of the individuals (>90%) exhibit a predominant IgG immune response to infection with H.pylori [19]. And approximately 70% of these individuals also exhibit IgA antibodies. However some investigators [15,20] have found that about 2% of the patient produces an IgA response in absence of IgG response.

In our population we found 7% (16/228) of IgA- positive response and IgG –negative response. Jaskowski et al., [21] also showed higher frequency (7.2%) of IgA -positive and IgG -negative patients. Luthra et al., [19] also states that approximately 7% of infected individuals are positive for IgA antibodies but negative for IgG antibodies; the reason for this aberrant response remains unclear.

IgA response was more common in Duodenitis patients and reflux esophagitis patients and least in duodenal ulcer patients. On the contrary Granberg et al., [15] establish the importance of IgA, as an increase risk factor of peptic ulcer disease and gastric cancer. Kosunen et al., [22] also showed the prevalence of IgA antibody highest in gastric ulcer patients, second highest in duodenal ulcer and chronic gastritis and are associated with the serious sequel of H.pylori infection.

EIA-G update is reliable and accurate test and because of its 100% sensitivity and negative predictive values, makes it useful screening test and thus serve as an alternative to endoscopy. Great care is to be taken not to underestimate the prevalence of H.pylori infection from the results of IgG serology in clinical practice. A positive finding of IgA antibody may be of significant clinical value in supporting diagnosis of infection especially if IgG serology is negative.

We are indebted to Dr. Parimal N. Salvi (Gastroenterologist) for providing us the Biopsy specimens for carrying out the Gold standard tests for H.pylori.

[1]. A Van de Wouw B, A de Boer W, R Jansz A, T Roymans R, A Staals, Comparison of three commercially available enzyme-linked immunosorbent assays and biopsy-dependent diagnosis for detecting Helicobacter pylori infection Journal of Clinical Microbiology 1996 34:94-7.  [Google Scholar]

[2]. HO Bow, BJ Marshall, Accurate Diagnosis of Helicobacter pylori: Serologic Testing. Gastroenterology clinic of North America 2000 29:853-62.  [Google Scholar]

[3]. RA Salomaa, TU Kosunen, J Mattila, S Sarna, H Rautelin, Age-dependent accuracy of Helicobacter pylori antibody assays for adults, with special emphasis on atrophic gastritis. Clin Diagn Lab Immunol 2004 11:1185-8.  [Google Scholar]

[4]. M Mohammadi, Y Talebkhan, G Khalili, F Mahboudi, S Massarrat, Zamaninia 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.  [Google Scholar]

[5]. D Vijaya, N Chandrashekhar, T Nagarantnamma, AS shivarudrappa, Simple stain for Helicobacter pylori. J clin diagn Res 2012 6(4):664-6.  [Google Scholar]

[6]. M Yousuf, UA Rao, SP Thyagarajan, A comparative study between rapid urease [Modified], CLO-test, culture & histopathological examination for Helicobacter pylori in-patients with acid peptic disease. Indian J Pathol Microbiol 1995 38(4):349-54.  [Google Scholar]

[7]. V Destura R, D Labio E, J Barrett L, S Alcantara C, I Gloria V, L Daez M, Laboratory diagnosis and susceptibility profile of Helicobacter pylori infection in the Philippines. Annals of clinical microbiology and antimicrobials 2004 3(25)  [Google Scholar]

[8]. O Rotimi, A Cairns, S Gray, Histological identification of Helicobacter pylori: comparison of staining methods J Clin Pathol. 2000 53:756-9.  [Google Scholar]

[9]. S Mahmood, A Hamid, Comparison between Invasive and Noninvasive Tests in Diagnosis of Helicobacter pylori Infection Pakistan Journal of Biological Sciences 2010 13:509-12.  [Google Scholar]

[10]. JC Atherton, Non-endoscopic tests in the diagnosis of Helicobacter pylori infection Aliment Pharmacol Ther. 1997 11(1):11-20.  [Google Scholar]

[11]. Y Urita, K Hike, N Torii, Y Kikuchi, H Kurakata, E Kanda, Comparison of Serum IgA and IgG Antibodies for Detecting Helicobacter pylori Infection. Internal Medicine 2004 43:548-52.  [Google Scholar]

[12]. Z Rahman H, G Azam M, A Rahman M, S Arfin M, M Alam, B Tareq M, Non-invasive diagnosis of H.pylori infection: Evaluation of serological tests with and without current infection marker CIM. World J Gastroenterol 2008 14(8):1231-6.  [Google Scholar]

[13]. RC She, AR Wilson, CM Litwin, Evaluation of Helicobacter pylori Immunoglobulin G (IgG), IgA, and IgM Serologic Testing Compared to Stool Antigen Testing. Vaccine Immunol 2009 16(8):1253  [Google Scholar]

[14]. A Locatelli, WR Catapani, Junior Gomes CR, C Battistin, P Silva, Detection of anti-Helicobacter pylori antibodies in serum and duodenal fluid in peptic gastroduodenal disease. World J Gastroenterol 2004 10(20):2997-3000.  [Google Scholar]

[15]. Granberg C, Mansikka A, Lehtonen OP, Kujari H, Grönfors R, Nurmi H, Diagnosis of Helicobacter pylori infection by using PYLORISET ELISA-G and ELISA-A for the detection of serum immunoglobulin G [IgG] and IgA antibodies. J Clin Microbiol. 1993 31(6):1450  [Google Scholar]

[16]. DG Newell, BL Johnston, H Ali M, PI Reed, An enzyme-linked immunosorbent assay for the serodiagnosis of Campylobacter pylori-associated gastritis. Scand J Gastro-enterol. 1988 23(142):53-7.  [Google Scholar]

[17]. TU Westblom, JS Barthel, TU Kosunen, ED Everett, Serum IgG and IgA antibody responses to Campylobacter pylori in a group of healthy asymptomatic volunteers Scand J Infect Dis 1989 21:311-4.  [Google Scholar]

[18]. JI Wyatt, Histopathology of gastro duodenal inflammation: the impact of Helicobacter pylori Histopathology. 1995 26(1):15  [Google Scholar]

[19]. G Luthra, AR Di Nuzzo, WK Gourley, SE Crowe,, Prevalence of Helicobacter pylori infection in patients undergoing endoscopy: Comparison of biopsy and serological methods of diagnosis and role of antibiotics American Journal of Gastroenterology 1998 93:1291-6.  [Google Scholar]

[20]. TU Kosunen, K Seppala, S Sarna, P Sipponen, Diagnostic value of decreasing IgG, IgA and IgM titres after eradication of Helicobacter pylori Lancet. 1992 339:893-5.  [Google Scholar]

[21]. TD Jaskowski, TB Martins, HR Hill, CM Litwin, Immunoglobulin A antibodies to Helicobacter pylori. J Clin Microbiol 1997 35:2999-3000.  [Google Scholar]

[22]. TU Kosunen, A Aromaa, P Knekt, Helicobacter antibodies in 1973 and 1994 in the adult population of Vammala, Finland Epidemiol Infect 1997 119:29-34.  [Google Scholar]