Comparison of chemiluminiscence versus lateral flow assay for the detection of Helicobacter pylori antigen in human fecal samples

Helicobacter pylori is a Gram-negative bacterium that causes chronic gastric inflammation, which can lead to gastric neoplasia. Therefore, early diagnosis of H. pylori infection is crucial for effective treatment and prevention of complications. The aim of this study was to compare the sensitivity and specificity of the STANDARD™ F H. pylori Ag FIA stool antigen test (SD Biosensor) with the LIAISON® Meridian H. pylori SA for the diagnosis of H. pylori infection. A total of 133 stool samples from patients with suspected H. pylori infection were compared using the STANDARD™ F H. pylori Ag FIA stool antigen test (SD Biosensor), based on lateral flow assay, with the LIAISON® Meridian H. pylori SA. Of the 45 positive samples with LIAISON, 44 were also positive while 1 was negative in the STANDARD™ antigen test. However, this discrepant sample showed a chemiluminescence index of 1.18, very close to the cut-off point of 1. On the other hand, of 88 negative samples obtained with LIAISON, 83 were negative and 5 were positive in the STANDARD™ antigen test. Moreover, STANDARD™ F H. pylori Ag FIA assay has shown a sensitivity of 97.8% (95% CI: 88.2-99.9), a specificity of 94.3% (95% CI: 87.2-98.1), a PPV of 83.9% (95% CI: 68.9-92.4) and a NPV of 99.3% ((95% CI: 95.3-99.9). In conclusion, the STANDARD™ F H. pylori Ag FIA (SD Biosensor) on the STANDARD™ F2400 analyser is a highly sensitive, specific and suitable assay for the detection of H. pylori in stool samples.


Introduction
Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic demanding, spirophilic, highly motile bacillus that colonizes the human gastric epithelium [1]. It is transmitted mainly by the fecal-oral and possibly by the oral-oral route [2]. Despite the decreasing prevalence of H. pylori infection, this bacterium still infects 30-50% of the general population in developed countries [3]. In addition, the development of H. pylori infection is related to the host conditions, the socioeconomic factors and the sanitary conditions. Consequently, the prevalence varies according to the geographical area and the level of development of different countries [1].
This microorganism can cause inflammation of the gastric mucosa, which in some cases can lead to complications such as dyspepsia, peptic ulcer, gastric malignancies and extragastric diseases [3]. Likewise, the World Health Organization detected 812,000 new cases of gastric malignancy attributable to H. pylori in 2018, being the third leading cause of cancer death [4].
The H. pylori detection techniques include both invasive (endoscopy, gastric tissue biopsy culture, gastric tissue polymerase chain reaction (PCR), rapid urease test and biopsy histology) and non-invasive (serology, stool antigen test and urea breath test) [5]. The stool antigen tests consist of a qualitative technique based on enzyme-linked immunoassay Carlos Rescalvo-Casas and Marcos Hernando-Gozalo contributed equally to this study. (ELISA) or lateral flow assay that detects the presence of H. pylori antigen [1]. In addition, they have a sensitivity and specificity greater than 95% and they are reliable, simple, rapid and inexpensive [6]. The sensitivity and the specificity data vary between the different tests used. In recent metaanalyses, it was observed that these data correspond to a 91.4-92.4% in sensitivity and 91.9-93.0% in specificity [7]. Therefore, they constitute a good tool for the diagnosis of this infection, especially in the Primary Care area [1]. However, the stool antigen test can be falsely negative if there are a low density of H. pylori in the stomach and a low antigen load in the stool, which may be caused by the use of bismuth, proton pump inhibitor or antimicrobials, unformed or watery stool samples, and the time interval after eradication. In addition, the temperature and the interval between stool sample collection and measurement also affect the stool antigen test results [7].
In summary, an early diagnosis of this infection is essential in order to establish an effective treatment in patients and thus avoid the complications mentioned above. Consequently, the aim of this study was to compare the sensitivity and the specificity for the detection of H. pylori in human fecal samples, using STANDARD TM F H. pylori Ag FIA (SD Biosensor) on the STANDARD TM F2400 analyzer or the LIAISON® Meridian H. pylori SA (DiaSorin) on the LIAISON® XL platform, which was considered the reference standard.

Material and Methods
A total of 133 human fecal samples from different patients suspected of Helicobacter pylori infection have been prospectively analyzed during three non-consecutive days and studied in parallel with STANDARD TM F H. pylori Ag FIA (SD Biosensor Inc, Suwon, South Korea) and LIAISON® Meridian H. pylori SA (DiaSorin Inc, Minneapolis, Minnesota USA) assays. For the analysis of the results, the LIAI-SON® Meridian H. pylori SA has been taken as the reference assay, and the results obtained with this assay have been considered the true results. 48 samples were positive and 85 were negative for LIAISON® Meridian H. pylori SA.
The samples were processed according to the instructions provided by each company. Briefly, a sufficient amount of the stool samples was collected using a swab. The content of the swab was mixed with the commercial dilution buffer found in the test. Three drops of the mixture were then added at 30-second intervals onto the immunochromatography strip. This was introduced into the STANDARD TM F2400 analyser, reading the obtained results after 10 minutes. The samples were evaluated in parallel by LIAISON® Meridian H. pylori SA. In the cases where there was a discordant result between the two antigen detection tests, the Allplex™ H. pylori/ClariR PCR (Seegene Inc. Seoul, South Korea) was performed.
The statistical analysis was performed with Stata / IC 13.1 (StataCorp, Texas, USA). The 95% confidence interval (95% CI) values were calculated using the Wilson method. The degree of agreement between the reference technique (LIAISON) and the evaluated test was determined with Cohen's Kappa index. Positive predictive (PPV) and negative predictive values (NPV) were obtained according to the prevalence of H. pylori infection in our population.

Results
The prevalence of positives in our population has been 23.2%, out of 11,596 samples analysed between 2018-2022.
The obtained results of positive or negative test of the LIAISON® and SD BIOSENSOR are shown in the Table 1.
The sensitivity, specificity and positive or negative predictive value obtained in the STANDARD™ F H. pylori Ag FIA (SD Biosensor) test are shown on Table 2.
In addition, these tests showed an agreement of 95.0% and a Cohen's Kappa index of 0.901 (almost perfect agreement).
Two negative samples of Diasorin LIAISON were positive in SD BIOSENSOR. PCR was performed to confirm these results, establishing that they were negative.

Discussion
In this study, the sensitivity and specificity of the STAND-ARD™ F H. pylori Ag FIA (SD Biosensor) test for the determination of H. pylori infection were compared. The  [9]. Studies similar to ours using the Cohen Kappa index, showed a similarity of 0.885 [10]. The accuracy of monoclonal stool antigen test for the diagnosis of H. pylori infection was assessed in the systematic review and meta-analysis of Gisbert J.P. et al. [11]. The results of their study suited that monoclonal antigenic tests have a higher sensitivity than polyclonal tests, especially in post-treatment measure [11]. In our study, five false positives were obtained by the antigenic tests. The reasons for these failures could be various, such as temperature-related reasons [7,10], watery stool samples [7], time lag between sample collection and measurement [7], poor storage of reagents [10] or heterogeneous distribution of antigens [10].
In addition, for invasive techniques, specific equipment and qualified personnel are needed to be able to take a good sample and perform the test well, whereas for noninvasive techniques, the procedure is simpler and faster. In the past, the specificity and sensitivity of non-invasive techniques could not compete with invasive techniques. However, improved PCR technology and antigenic tests are approaching the same results as invasive techniques. In the future, these techniques could replace biopsies for the detection of H. pylori infection [7].
These new and faster techniques could have a very similar diagnostic value to the invasive ones [7] or other non-invasive screening techniques [10]. Moreover, due to the high prevalence of H. pylori infection worldwide, this technique could show a high NPV, thus aiding to rule out infection.

Conclusions
The diagnostic performance of STANDARD™ F H. pylori Ag FIA (SD Biosensor) has proved to be an alternative to the chemiluminiscence antigenic test used in our population.
The STANDARD™ F H. pylori Ag FIA (SD Biosensor) on the STANDARD™ F2400 analyzer is a highly sensitive, specific and suitable assay for the detection of H. pylori in stool samples. The results have shown almost perfect concordance with the LIAISON® H. pylori SA assay. In addition, it also saves sample handling and working time as no sample pretreatment is required.
Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. C.R.-C and M. H.-G. acknowledge to the University of Alcala for their fellowships (FPU and FPI 2021, respectively). This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Data availability All data generated or analyzed during this study are included in this manuscript.

Declarations
Ethical approval The study was conducted according to the ethical requirements established by the Declaration of Helsinki. The Ethics Committee of Hospital Universitario Príncipe de Asturias (Madrid) approved the study.
Informed consent Informed consent waiver was authorized by the Ethics Committee.

Conflict of interest
The authors declare that they have no conflicts of interest.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.