Serum Anti-Helicobacter pylori IgG Antibody Titer in H. pylori-negative Cases with a Different Gastric Mucosal Atrophy Status.

Objective This retrospective study was performed to investigate the anti-Helicobacter pylori IgG antibody serum titers in H. pylori-negative subjects with different degrees of gastric mucosal atrophy including C0 grade atrophy. Methods The absence of H. pylori infection was determined based on both negative serum anti-H. pylori IgG antibody test findings and no endoscopic evidence of that infection. Cases negative for the antibody and with positive endoscopic findings of H. pylori infection were defined as H. pylori-positive. The serum anti-H. pylori IgG antibody titers were analyzed in H. pylori-negative (n=1,087), -positive (n=69), and post-eradicated (n=278) subjects. Results The serum antibody titer in subjects with H. pylori-positive endoscopy findings was significantly higher than that in H. pylori-negative subjects, even when the serum titer indicated a negative result. In addition, the anti-H. pylori IgG antibody serum titer was higher in H. pylori-negative subjects with a greater degree of gastric mucosal atrophy. In a comparison between H. pylori-negative C0 and C1 gastric mucosal atrophy cases, the antibody serum titer in those classified as C0 was significantly lower. An analysis of H. pylori post-eradicated cases showed that the serum antibody titer decreased over time after successful eradication. Conclusion The disappearance of H. pylori infection in H. pylori-negative individuals may occur later in those with a greater degree of gastric mucosal atrophy. The serum antibody titer difference between the H. pylori-negative C0 and C1 groups might have been caused by the differences in distribution between H. pylori-uninfected subjects and those in whom the infection had disappeared, thus additional investigation is needed to clarify the significance of gastric mucosal classification including the C0 grade.

Introduction

A long-term Helicobacter pylori infection is the most important factor for the development of gastric mucosal atrophy (1-3). However, some individuals show apparent endoscopic gastric mucosal atrophy without that infection, even though they have not undergone eradication therapy (4, 5). This condition is considered to be caused by the spontaneous disappearance of H. pylori or the usage of an antibiotics for another condition (6-9). H. pylori infection generally initiates in childhood, while the degree of gastric mucosal atrophy gradually increases with age (10, 11). Therefore, the timing of the disappearance of that infection is expected to have an effect on degree of gastric mucosal atrophy in H. pylori-negative individuals. Since the serum titer of the anti-H. pylori IgG antibody has been reported to decrease after the disappearance of infection by eradication therapy (12, 13), the timing of infection disappearance may also affect that serum titer. However, the relationship between gastric mucosal atrophy degree and anti-H. pylori IgG antibody serum titer has not yet been investigated in H. pylori-negative subjects who have no history of eradication therapy.

Endoscopic gastric mucosal atrophy classification was developed by Kimura and Takemoto, and it is based on 6 grades (C1, C2, C3, O1, O2, O3) (14). In addition, cases without gastric mucosal atrophy have recently been referred to as C0 grade (4), thus the classification of gastric mucosal atrophy can be divided into 7 groups, including the C0 grade and those noted above. To the best of our knowledge, the significance of endoscopic gastric mucosal classification that includes the C0 grade has not yet been determined. We conducted this retrospective study to clarify the relationship between serum titer of the anti-H. pylori IgG antibody and degree of gastric mucosal atrophy in subjects without H. pylori infection, as well as the significance of that classification with C0 grade included.

Materials and Methods

The subjects were selected from individuals who visited the Health Center of Shimane Environment and Health Public Corporation for a detailed medical checkup examination between June 2016 and November 2019, as we have been evaluating patients based on the endoscopic gastric mucosal atrophic classification including C0 grade since June 2016. The majority were socially active and productive, and considered to be socioeconomically middle class. During the study period, a total of 2,288 individuals were simultaneously assessed by both an upper GI endoscopic examination and serum anti-H. pylori IgG antibody test. Those with a history of gastric surgery were excluded from the present analysis. Of the 282 individuals with an H. pylori post-eradication status, 4 were excluded prior to the analysis, because the timing of eradication could not be determined. Thus, the number of post-eradicated cases subjected to analyze was 278 (post-eradicated group: 184 males, 94 females; mean age 55.0 years). Serum anti-H. pylori IgG antibody detection was performed using the SphereLight H. pylori antibody JⓇ kit (FUJIFILM Wako Pure Chemical, Osaka, Japan), originally developed as a qualitative test for the diagnosis of H. pylori infection, and the antibody titer was automatically determined using a chemiluminescent enzyme immunoassay method, with a value ≥4.0 U/mL defined as positive, according to the manufacturer's instructions (4, 15). The presence or absence of H. pylori infection, and the result of eradication therapy were also confirmed based on findings obtained in an upper GI endoscopic examination (16-20). In order to examine the serum titer of H. pylori-negative subjects, we excluded those found positive in a test for the anti-H. pylori antibody in serum. Sixty-nine subjects negative for the anti-H. pylori IgG antibody were placed in the H. pylori-positive group (47 males, 22 females; mean age 48.8 years), as they had endoscopic findings indicating the H. pylori-positive status, such as nodular gastritis, spotty and/or diffuse redness of fundic gland mucosa, or sticky mucous (16-20). The remaining 1,087 (606 males, 481 females; mean age 49.5 years) were placed in the H. pylori-negative group. The total number of subjects in this study was 1,434 (837 males, 597 females; mean age 50.5 years) (Fig. 1).

Figure 1.

Flow diagram of the subject selection process.

All upper GI endoscopic examinations were performed by experienced licensed endoscopists using an EG-L580NW endoscope (Fujifilm, Tokyo, Japan). At our institution, an upper GI endoscopic examination is performed with the subject in an unsedated condition without anti-cholinergic drug administration, with the endoscope typically inserted in a transnasal manner. When gastric mucosal atrophy was endoscopically observed, the degree was evaluated using the classification of Kimura and Takemoto, in which gastric mucosal atrophy is classified into 6 grades (C1, C2, C3, O1, O2, O3) (14). The degree of gastric mucosal atrophy was carefully determined by recognition of an atrophic border. Cases without gastric mucosal atrophy were determined to be C0 for this study. Therefore, subjects with C1 atrophy according to the original Kimura and Takemoto classification were classified as C0 or C1 (Fig. 2, 3), while gastric mucosal atrophy was classified into 7 grades, including C0 and the others noted above. All endoscopic images from each subject were simultaneously reviewed by three expert endoscopists to determine the degree of gastric mucosal atrophy and the absence of endoscopic findings indicating positive for H. pylori infection was confirmed by consensus.

Figure 2.

Representative endoscopic images showing C0 grade gastric mucosal atrophy. No atrophic mucosal area can be seen in the antrum with ordinary imaging (a) or linked color imaging (LCI) (b).

Figure 3.

Representative endoscopic findings demonstrating C1 grade gastric mucosal atrophy. An atrophic border is shown as differences in color in the antrum by ordinary imaging (a, b) and linked color imaging (LCI) (c). The atrophic area and border were easily recognized and visualized by LCI.

The serum anti-H. pylori IgG antibody titers in H. pylori-negative subjects with different degrees of gastric mucosal atrophy were analyzed and compared. The subjects with gastric mucosal atrophy graded from C3-O3 were combined into a single group for the present analysis, since the number with C3, O1, O2 and O3 atrophy was small (19, 3, 6, 13, respectively). In addition, the serum antibody titers were analyzed in subjects with H. pylori-positive endoscopic findings. In post-eradicated subjects, the serum antibody titers were compared after dividing them based on the duration after eradication to examine the relationship between the degree of gastric mucosal atrophy and age at time of successful eradication.

Statistical analyses were performed using a chi-squared test, Mann-Whitney U test, or the Kruskal-Wallis test, as appropriate. All calculations were done using the StatView 5.0 software program for Macintosh (Abacus Concepts, Berkeley, USA), with a p level <0.05 considered to indicate statistical significance.

This study was performed in accordance with the Declaration of Helsinki, and the protocol was approved by the ethics committee of the Shimane Environment and Health Public Corporation. Written informed consent indicating that obtained clinical data would be used for a clinical study without release of individual information was received from all subjects before performing the medical checkup examinations.

Results

The characteristics of the present H. pylori-negative and -positive subjects are shown in Table 1. The numbers of negative subjects with C0, C1, C2, and C3-O3 grade gastric mucosal atrophy were 716, 172, 158 and 41, respectively. Further analysis showed a female predominance in those with C2 atrophy, while those in the C3-O3 group were older in comparison to the others.

Table 1.

Characteristics of the H. Pylori-negative and -positive Subjects.

Gastric mucosal atrophy grade	H. pylori-negative (n=1,087)				H. pylori-positive
	C0 (n=716)	C1 (n=172)	C2 (n=158)	C3-O3 (n=41)	(n=69)
Gender, male/female	420/296	95/77	68/90#1, 2	23/18#3	47/22#3
Age, years	49.3±0.4	47.6±0.8#1	49.1±0.8	62.0±1.4#1, 2, 3	48.8±1.3#4
Serum titer of anti-H. pylori IgG antibody (U/mL)	0.81±0.02	0.99±0.06#1	1.07±0.07#1	1.54±0.13#1, 2, 3	2.63±0.12#1, 2, 3, 4
≤0.9	526 (73.5%)	112 (65.1%)	88 (55.7%)	13 (31.7%)	5 (7.3%)
1.0-1.9	152 (21.2%)	40 (23.3%)	47 (29.7%)	16 (39.0%)	12 (17.4%)
2.0-2.9	25 (3.5%)	15 (8.7%)	18 (11.4%)	10 (24.4%)	21 (30.4%)
3.0-3.9	13 (1.8%)	7 (2.9%)	5 (3.2%)	2 (4.9%)	31 (44.9%)

H. pylori-negative: negative in anti-H. pylori antibody test results and absence of H. pylori infection in endoscopic findings. H. pylori-positive: negative in anti-H. pylori antibody test results and positive for H. pylori infection in endoscopic findings. Values are expressed as the mean±SE or number of subjects. Gastric mucosal atrophy was evaluated using the modified classification of Kimura and Takemoto. #1, 2, 3, 4: significantly different in comparison to subjects with C0, C1, C2, C3-O3 grade, respectively.

The anti-H. pylori IgG antibody serum titer was greater in H. pylori-negative subjects with a higher degree of gastric mucosal atrophy. The serum titer levels were divided into four subgroups (<0.9, 1.0-1.9, 2.0-2.9, 3.0-3.9 U/mL) and analysis in H. pylori-negative subjects showed that the anti-H. pylori IgG antibody titer was significantly different among the gastric mucosal atrophy grades (p<0.001). Furthermore, when the anti-H. pylori IgG antibody titer was examined in H. pylori-negative subjects with C0 and C1 gastric mucosal atrophy, it was found to be significantly lower in subjects with the C0 grade, while the distribution of the serum titers was also significantly different between C0 and C1 (p=0.013). The anti-H. pylori IgG antibody titer in H. pylori-positive subjects was significantly higher than that in the H. pylori-negative subjects, while the antibody titer was also more largely distributed from 2.0-3.9 in the positive group (Table 1, Fig. 4).

Figure 4.

The serum titers of anti-H. pylori IgG antibody in H. pylori-negative, -positive, and post-eradicated cases. H. pylori-negative: negative in anti-H. pylori antibody test results and absence of H. pylori infection in endoscopic findings. H. pylori-positive: negative in anti-H. pylori antibody test results and positive for H. pylori infection in endoscopic findings. The numbers of H. pylori-negative, -positive, and post-eradicated subjects were 1,087, 69, and 278, respectively. Values are expressed as the mean±SE.

The further analyze the anti-H. pylori IgG antibody serum titer, H. pylori-negative subjects were divided based on gender, and older age and higher level of serum titer were noted in both males and females with a higher degree of gastric mucosal atrophy. Furthermore, the distribution of the anti-H. pylori IgG antibody titer was significantly different among the gastric mucosal atrophy grades in both genders (p<0.001). Also, a comparison between the C0 and C1 gastric mucosal atrophy groups showed that the anti-H. pylori IgG antibody titer tended to be lower in both males and females with C0 grade, with a similar tendency observed in the distribution of serum titers between the genders (Table 2).

Table 2.

Characteristics of the Subjects without H. Pylori Infection Divided by Gender.

Gastric mucosal atrophy grade	C0	C1	C2	C3-O3
Male
Age, years	50.0±0.5	48.2±1.1	50.0±1.2	63.2±1.9#1, 2, 3
Serum titer of anti-H. pylori IgG antibody (U/mL)	0.77±0.03	1.01±0.08#1	1.21±0.11#1	1.38±0.16#1, 2
≤0.9	324 (77.1%)	61 (64.2%)	32 (47.1%)	9 (39.1%)
1.0-1.9	77 (18.3%)	20 (21.1%)	23 (33.8%)	9 (39.1%)
2.0-2.9	12 (2.9%)	11 (11.6%)	9 (13.2%)	4 (17.4%)
3.0-3.9	7 (1.7%)	3 (3.2%)	4 (5.9%)	1 (4.3%)
Female
Age, years	48.3±0.6	46.9±1.2	48.5±1.0	60.4±2.1#1, 2, 3
Serum titer of anti-H. pylori IgG antibody, U/mL)	0.87±0.04	0.95±0.08#1	0.95±0.08	1.77±0.22#1, 2, 3
≤0.9	202 (68.2%)	51 (66.2%)	56 (62.2%)	4 (22.2%)
1.0-1.9	75 (25.3%)	20 (26.0%)	24 (26.7%)	7 (38.9%)
2.0-2.9	13 (4.4%)	4 (5.2%)	9 (10.0%)	6 (33.3%)
3.0-3.9	6 (2.0%)	2 (2.6%)	1 (1.1%)	1 (5.6%)

Data are expressed as the mean±SE or number of subjects. Gastric mucosal atrophy was evaluated using the modified classification of Kimura and Takemoto. #1, 2, 3: significantly different in comparison to subjects with C0, C1, C2 grade, respectively.

An analysis of the anti-H. pylori IgG antibody serum titer in the H. pylori-eradicated group showed that it decreased for a longer duration after successful eradication (Fig. 4). In addition, the degree of gastric mucosal atrophy when successful eradication was achieved was significantly lower in younger subjects (Table 3).

Table 3.

Relationship between the Age at Time of Eradication and the Degree of Gastric Mucosal Atrophy in H. Pylori Post-eradicated Cases.

	Degree of gastric mucosal atrophy (p < 0.001)
Age (years)	C1	C2	C3	O1	O2	O3
≤ 39 (n=44)	8 (18.2%)	24 (54.5%)	11 (25.0%)	1 (2.3%)	0	0
40-49 (n=97)	14 (14.4%)	37 (38.1%)	35 (36.1%)	8 (8.2%)	3 (3.1%)	0
50-59 (n=90)	8 (8.9%)	35 (38.9%)	25 (27.8%)	12 (13.3%)	5 (5.6%)	5 (5.6%)
≥60 (n=47)	1 (2.1%)	13 (27.7%)	8 (17.0%)	7 (14.9%)	9 (19.1%)	9 (19.1%)

Data are expressed as the number of subjects, unless otherwise noted. Gastric mucosal atrophy was evaluated using the classification of Kimura and Takemoto.

Discussion

In this study, we determined the anti-H. pylori IgG antibody titer in serum obtained from H. pylori-negative, -positive, and post-eradicated subjects. An analysis of H. pylori-negative subjects found a higher titer in those with a higher degree of gastric mucosal atrophy, while comparisons between subjects with C0 and C1 gastric mucosal atrophy also revealed a significant difference regarding that antibody titer. The timing of the disappearance of H. pylori infection is considered to affect not only the degree of gastric mucosal atrophy but also serum anti-H. pylori IgG antibody titer. The progression of gastric mucosal atrophy induced by such infection discontinues after successful eradication of the bacterium. Thus, the degree of gastric mucosal atrophy is considered to reflect that at the time of disappearance of infection, though improvement may actually occur quite slowly over a long period. Indeed, findings for the H. pylori-eradicated group demonstrated that the degree of gastric mucosal atrophy was significantly lower in those who were younger at the time of successful eradication. Other studies have noted that the anti-H. pylori IgG antibody titer in the serum was reduced after successful eradication (6, 7), while the present results also demonstrated that the titer in H. pylori-eradicated subjects had a greater decrease in those with a longer duration following eradication. Thus, a reduced titer in individuals with a milder degree of gastric mucosal atrophy is considered to be related to a long duration after the disappearance of infection. As a result, the disappearance of H. pylori infection in H. pylori-negative patients with a greater degree of gastric mucosal atrophy may occur later, though that timing could not to be determined in this study.

H. pylori-negative subjects did not have a current H. pylori infection during the test period, but they consisted of two types; those who had never been infected and those who had a previous infection that had disappeared prior to our examination. The disappearance of H. pylori can be caused by targeted eradication therapy, a spontaneous disappearance, or the usage of antibiotics for another disease condition (6-9). A spontaneous disappearance is considered to occur in individuals with severe gastric mucosal atrophy, as the bacterial organisms cannot survive in the mucosa of intestinal metaplasia induced with progression of the disease. Among the present study subjects without H. pylori infection, only 13 had an O3 grade of gastric mucosal atrophy, while the 186 with a grade ranging from C2-O2 showed a non-atrophic fundic gland area in endoscopic findings, in which H. pylori can survive. Thus, an H. pylori-negative status in individuals with gastric mucosal atrophy is likely not caused by disease progression, with the usage of antibiotics for another condition considered to be the main reason for bacterial negativity.

The present subjects without H. pylori infection were divided into those who had never been infected (uninfected), and those with an infection that occurred during childhood and then later disappeared for some reason (disappeared). Interestingly, the values for both the titer and distribution of the anti-H. pylori IgG antibody in serum were significantly different between the C0 and C1 groups in the uninfected subjects. In the subjects with C0 atrophy, the serum titer was lower and those subjects were also more predominantly represented in the group with a lower serum titer of the antibody in comparison with the C1 group. These findings suggest that the distribution of H. pylori-uninfected and disappeared subjects is different when divided between the C0 and C1 atrophy status.

The present H. pylori negative subjects with C3-O3 atrophy were older than those with milder atrophy. Patients with C3-O3 atrophy are considered to have been previously infected by H. pylori for a long period and its disappearance likely occurred later in comparison to those with a milder degree of gastric mucosal atrophy. A female predominance was also noted in the C2 atrophy group in comparison to the others, though our findings could not clearly explain this phenomenon. However, the values for the titer and distribution of the anti-H. pylori IgG antibody were not different between males and females without H. pylori infection. Gender difference in regard to the progression of gastric mucosal atrophy during H. pylori infection might have been a factor, since the events correlated with disappearance of H. pylori infection such as the usage of antibiotics for another condition were considered to be similar in the female and male subjects.

In this study, 69 (6.0%) of 1,156 subjects negative for the anti-H. pylori IgG antibody were diagnosed as being positive for H. pylori infection. Testing was done with the SphereLight H. pylori antibody JⓇ test, which has been demonstrated to have a high level of sensitivity (4, 15). We noted that the anti-H. pylori IgG antibody titer in the H. pylori-positive subjects was significantly higher than that in the negative subjects, and their results were more largely distributed near the cut-off value of this test. Endoscopic H. pylori-positive findings, including nodular gastritis, spotty and/or diffuse redness of fundic gland mucosa, and sticky mucous, were carefully reviewed simultaneously by three endoscopy experts. Although H. pylori infection was not examined by other diagnostic methods, the higher anti-H. pylori IgG antibody titer in subjects with endoscopic findings indicating positive for H. pylori infection is considered to indicate the importance of a detailed endoscopic examination in the cases found to be negative in a serum antibody test. Recently, patients with a non-Helicobacter pylori Helicobacter (NHPH) infection have been reported to have endoscopic findings of nodular gastritis and a white marbled appearance, which appeared to demonstrate atrophic change (21, 22). Therefore, some subjects in the present H. pylori-positive group may have had an NHPH infection. Additional investigations are needed to clarify the influence of NHPH infection on the endoscopic diagnostic findings indicating positive for H. pylori infection.

The present study is associated with several limitations. It was not performed in a population-based manner, as the subjects initially visited our center for a medical check-up. Also, the investigation was performed in a cross-sectional manner, thus we were unable to determine events that might have caused disappearance of H. pylori infection in the subjects. Furthermore, we did not perform histological examinations of the gastric mucosa samples obtained from the subjects, since the endoscopic examinations in this study were performed as part of a medical checkup. Thus, we could not obtain histological findings regarding the presence of NHPH, nor showing non-atrophic fundic or pyloric gland areas. In addition, we used the results from a single type of anti-H. pylori IgG antibody test and the results of different tests might not be identical to those found in this study. The present results should be confirmed by a future large-scale study that includes histological examinations and other serological testing methods in addition to the SphereLight H. pylori antibody JⓇ test.

In conclusion, the anti-H. pylori IgG antibody titer in serum in H. pylori-negative subjects with a higher degree of gastric mucosal atrophy was higher in comparison to those with a lower degree. We concluded that a later disappearance of H. pylori infection may occur in patients with a high degree of gastric mucosal atrophy. Additionally, a lower serum titer was seen in H. pylori-negative subjects with C0 atrophy and those subjects were more predominantly represented in the group with a lower serum titer of the antibody. Thus, the distribution of subjects classified as H. pylori-uninfected and disappeared may be different between those with a C0 and C1 atrophy status, and additional investigation is needed to clarify the significance of the gastric mucosal classification including the C0 grade.

The authors state that they have no Conflict of Interest (COI).