Comparison of rapid cassette test and micro ELISA method for Helicobacter pylori diagnosis in patients with gastric complaints

Background: Helicobacter pylori (H. pylori) is a significant contributor of various gastrointestinal disorders and cancers all around the world. Its diagnosis is dependent on several qualitative and quantitative methods. The present study aims to compare the results of rapid cassette and micro ELISA test methods for diagnosis of H. pylori and determining associations with patient endoscopy reports. Methods: The study was performed using blood samples collected from 224 patients (142 (63%) females and 82 (37%) males) in various clinics between January 2018 and August 2019, which were sent to the Clinical Microbiology Laboratory of Training Hospital. Serum samples obtained after centrifugation of the blood samples were initially tested with rapid H. pylori IgG cassette method, and afterwards in the auto analyzer using ELISA assays specific for H. pylori. Results: Upper gastrointestinal system endoscopy was performed in 88 of these patients, and biopsy results confirmed definitive diagnosis of H. pylori infection in 63 of the patients. Rapid H. pylori cassette test results of the 224 patients were negative for 158 (70.5%) patients and positive for 66 (29.5%) patients, whereas micro ELISA IgA test results were negative for 110 (49.1%) patients and positive for 114 (50.9%) patients. Micro ELISA IgG test results were negative for 85 (37.9%) patients and positive for 139 (62.1%) patients. Conclusions: Invasive diagnostic methods for H. pylori infection may sometimes be inconvenient, and therefore the diagnosis may have to rely on non-invasive tests. Bases on the study results, we believe micro ELISA test results are more reliable with regard to avoidance of missed diagnosis.

people living in socioeconomically poor environments and developing countries. Africa currently has the highest prevalence rates, which exceed 70 percent. The causes that lead to high prevalence in these countries are excessive population, insufficient housing, poor hygiene, and polluted water [7,8].
Infection is typically acquired during childhood; however, it stays latent for a long time and continues into adulthood. Therefore, infected individuals are usually unaware of the infection and tend to spread it to other people. Only a small portion of infected individuals develops disease in their adulthood [9].
In 1994, H. pylori was defined as a Group 1 carcinogenic agent by International Agency for Research on Cancer (IARC), and was associated with various other systemic disorders including hematological, skin, cardiovascular and respiratory diseases. Given various diagnostic methods and patient clinical conditions, high sensitivity methods should be preferred for diagnosis of the infection [10,11]. H. pylori infection can be detected with invasive or non-invasive methods. However, due to complications associated with the invasive tests, routine laboratory studies often employ non-invasive immunological assays for diagnosis. Commonly used non-invasive tests are urea breath test (UBT) and serological tests in serum, urine, or other body secretions. These include rapid cassette test (RCT), H. pylori stool antigen test (HpSA), and H. pylori ELISA assays [8][9][10][11][12]. Currently in the USA, serological test methods are the most commonly used diagnostic tools for H. pylori infections [13].
The present study aims to compare the results of H. pylori IgG cassette test with H. pylori IgG and IgA ELISA tests from blood samples of patients thought to have H. pylori infection.

Methods
Patient population: Patient samples were obtained from 224 patients, 142 (63.4%) females and 82 (36.6%) males, who presented to various clinics of Training Hospital due to gastric complaints and provided blood samples between January 2018 and August 2019 for testing H. pylori infection.
Specimen collection: Approximately 4 ml of blood samples were collected in serum separator tubes and centrifuged at 5,000 rpm for 10 minutes to obtain a serum sample. Serum samples were stored at -20°C until the time of analysis. Prior to analysis, the samples were brought to room temperature.
H. pylori IgG Cassette Test: The test was a double antigen chromatographic lateral flow immunoassay.
The kit package (H. pylori IgG Cassette Test, Biocare Diagnostic, PDI GmbH, Essen, Germany) included a cassette, which contains a nitrocellulose diaphragm, with control (C) and test (T) lines marked on the cassette. The T line was coated with H. pylori antigens, and the C line was coated with goat antibodies against H. pylori. The kit package was stored at room temperature until analysis.
After bringing the patient samples to room temperature, 0.2 ml serum (approximately 4 drops) was pipetted to the sample chamber on the cassette. If the patient sample has specific antibodies against H. pylori, they combine with the target antigens and cause a red band to appear at the T line. A red band appears at the C line regardless of the presence of antibodies in the patient sample. The result was obtained approximately 5-8 minutes after pipetting the sample. As recommended by the manufacturer, presence of red coloration at the C line, which indicates the validity of the test result, and lack of red coloration at the T line was interpreted as "negative" result, while presence of red coloration at both lines was interpreted as a "positive" test result ( Figure 1).
H. pylori ELISA IgG / IgA: These tests are quantitative indirect immunoassays that measures specific IgG or IgA type antibodies against H. pylori in human serum or plasma, and it is based on the reaction between antigens adsorbed to the polystyrene surface and antibodies present in the tested sample. In this method, after removing unbound antibodies through a washing step, the antigen-antibody complex was bound to anti-human globulin marked with an enzyme. After a second washing step, an acid stop solution was added, and bound conjugate was added with the help of a substrate to obtain blue colored product that turns to yellow. The kit package (H. pylori ELISA IgG or IgA, Vircell MICROBIOLOGISTS, Granada, Spain) contained a 96 well plate coated with H. pylori 26695 strain antigens that were soluble in detergent, 25 ml serum diluent, positive and negative controls, conjugate, substrate, and washing solution.
The kit was stored at 4 C until time of analysis. As recommended by the manufacturer, reagents and solutions supplied with the kit were prepared and loaded to an automated device (Triturus ELISA Instrument, Grifols, Barcelona, Spain), and all the steps described above were performed automatically. Antibody index was calculated using the formula: sample optical densities (O. D.)/cut off serum mean O. D.) X 10. Accordingly, the results were interpreted as "negative" if the index was <9, "borderline" if index was between 9-11, and "positive" if the index was >11. H. pylori ELISA IgG and IgA results were reported separately following an analysis time of approximately 4 hours.
Upper GIS Endoscopy report: A total of 88 patients were examined with endoscopy. Endoscopic examination was performed following premedication procedure, using an appropriate gastroscope (Fujinon EG-600 WR, Fujifilm EU, Germany) and a digital image transfer system (Medgate 2000, Aort, Turkey). Esophagus, stomach and duodenum were examined. In case of observation of suspicious lesions associated with H. pylori infection, two biopsies were obtained from each of gastric corpus and antrum regions and were sent to pathology laboratory for histological examination. Pathology results were categorized as either positive or negative for H. pylori. In this study, informed consent was obtained from all participants, as written, before upper gastrointestinal endoscopy and biopsy procedure.

Results
Of the 224 patients included in the study, 142 (63.4%) were female and 82 (36.6%) were male. In addition, they applied a questionnaire to assess the socioeconomic levels and life styles of their patients. Accordingly, smoking, poor sanitation and lack of formal education were found as predisposing factors to infection (p<0.05). The two methods yielded the same results in 87.9% of the patients. In our study, we rather measured serum samples with three serological methods, and we found prevalence rates as 29.5% (66/224) with IgG cassette test, 50.9% (114/224) with ELISA IgA test and 62.1% (139/224) with ELISA IgG test. As observed, prevalence rates were somewhat higher with ELISA methods. This may be related to the selection of the patient group that we included in this study.
In a 2013-14 study with 160 students, Yo et al. [11] obtained 0.5 ml of saliva samples and analyzed them within 5 minutes using a H. pylori Saliva Test Cassette. They found positive results in 82 of the subjects and negative results in 78, and calculated the oral H. pylori infection rate as 51%. In addition, the subjects were questioned in terms of smoking, dietary and dental care habits and family history. Of the 82 subjects with positive test results, 74 had poor dental care. We did not apply the questionnaire to our patients; however, the results we obtained from the serological tests (positive results for IgG cassette, ELISA IgA, and ELISA IgG were 54 (61.4%), 66 (75.0%) and 68 (77.3%), respectively) were consistent with the findings of the aforementioned study.
Agbor et al. [18] conducted a prevalence analysis to determine the epidemiological profile of H. pylori infection by obtaining blood and stool samples from 500 patients with gastric complaints between 2013 and 2015. They used a one-step H. pylori antibody device for serum analysis. Three drops of serum was put into a well in the device and the result was obtained after 10 minutes. For stool analysis, a one-step H. pylori antigen test device was also used; 50 mg solid or two drops of watery stool sample was transferred to a tube and mixed with buffer. Two drops of this mixture was put into a well in the device and the result was obtained after 10 minutes. Of the 500 stool samples examined, 237 (43%) were positive for the H. pylori antibody test. Seropositivity rates were found to be similar between females and males (42% and 45%, respectively). They calculated the sensitivity and specificity of the antibody test (90% and 98%, respectively) in reference to the antigen test, which yielded higher positive rates. Twenty four samples were positive with the antigen test but negative with the antibody test. In contrast, 4 samples were positive with the antibody test but negative with the antigen test. Both tests yielded positive results for 213 of the samples. As a result, the authors noted that there was no significant difference between the two test methods (p = 0.204). In our study, we did not measure antigens in the stool; however, we used three different antibody tests and compared the results to the endoscopy results. The positivity rates that we obtained from the antibody tests were close to the results of the study mentioned above: 29. 5% In another study, Veijola et al.
[21] recruited their study participants via a newspaper advertisement, and included 1,574 adult subjects that did not receive antibiotic treatment for the last 2 months, or H2-receptor antagonists or bismuth or proton pump inhibitors for the last 2 weeks, or receive H. pylori eradication treatment for the last 5 years, or have history of gastric operation, chronic GIS disease, pregnancy or lactation. The subjects were tested with rapid whole blood antibody (IgG) test for diagnosis of H. pylori infection, and the 300 subjects with positive test results were confirmed with UBT and an in-house EIA-based serological assay (IgG and IgA). Of the 300 subjects, 196 were confirmed positive with both methods; however, since 11 subjects did not meet the inclusion criteria, 185 positive subjects were left. With the addition of 97 subjects who had positive results from confirmatory tests despite having negative screening test results, the total number of subjects enrolled in the study was 282 (186 females and 96 males). One hundred eighty five subjects who had positive results from all three methods were enrolled in the eradication program, and they were retested after 4 months with serological methods. The success criterion for eradication therapy was defined as at least 40% reduction in IgG antibody level. The performance of the three stool antigen tests, HpSA (polyclonal antibody-based), HpStAR (mAb-based Amplified IDEIA) and ImmunoCard (based on monoclonal H. pylori antibody and a lateral flow chromatography technique), which were applied to the subjects both before and after the eradication treatment, was evaluated in comparison to UBT and serology. Accordingly, pre-eradication sensitivity, specificity, PPV  In the study of Abu Shady [22], 100 pediatric patients with an age range of 4-10 years, who were referred to endoscopic examination due to upper GIS complaints were tested with rapid urease test (RUT) and biopsied for histological examination. For RUT, the result was obtained after adding biopsy sample to the urea solution (NaCl, KH2PO4 and NaOH). A change in the color of the urea solution from yellow to red, due to increase in pH induced by H. pylori, was accepted as a positive test result.
Histological examination was performed after staining with hematoxylin and eosin. Additionally, a microplate enzyme immunoassay (EIA) and an antibody detection kit were used to detect antibodies against H. pylori (IgG) in patient serum samples. The analysis was performed per manufacturer's instructions and cutoff threshold was 10U/mL The gold standard for diagnosis of H. pylori infection was accepted as positive results from both histological examination and the rapid urease test.
Accordingly, while standard test result was positive in 57% and negative in 43% of patients, serological test was positive in 60% and negative in 40% of patients. In addition, sensitivity and specificity values of anti-H. pylori IgG antibody test were found as 96.5% and 93%, respectively. The authors concluded that IgG antibody test was a good alternative to invasive diagnostic tests such as urea breath test, for diagnosis of H. pylori infection, and that IgG type of antibodies produced against H.pylori had higher diagnostic value. In contrast to that study, our study included adult patients.
Although the study designs were similar, we had lower sensitivity and specificity values. This was due to the fact that our study sample did not include children.

Conclusions
Even though it is accepted as the gold standard, invasive diagnostic methods for H. pylori infection may sometimes be inconvenient, and therefore the diagnosis may have to rely on non-invasive tests such as ELISA method and rapid diagnostic tests.    Figure 1 Evaluation of H. pylori IgG test results using rapid diagnostic tests in a group of patients.
The sample numbered as 14 appears to have a clearly positive test result.