Validation of a Novel Immunoline Assay for Patient Stratification according to Virulence of the Infecting Helicobacter pylori Strain and Eradication Status

Abstract

Helicobacter pylori infection shows a worldwide prevalence of around 50%. However, only a minority of infected individuals develop clinical symptoms or diseases. The presence of H. pylori virulence factors, such as CagA and VacA, has been associated with disease development, but assessment of virulence factor presence requires gastric biopsies. Here, we evaluate the H. pylori recomLine test for risk stratification of infected patients by comparing the test score and immune recognition of type I or type II strains defined by the virulence factors CagA, VacA, GroEL, UreA, HcpC, and gGT with patient's disease status according to histology. Moreover, the immune responses of eradicated individuals from two different populations were analysed. Their immune response frequencies and intensities against all antigens except CagA declined below the detection limit. CagA was particularly long lasting in both independent populations. An isolated CagA band often represents past eradication with a likelihood of 88.7%. In addition, a high recomLine score was significantly associated with high-grade gastritis, atrophy, intestinal metaplasia, and gastric cancer. Thus, the recomLine is a sensitive and specific noninvasive test for detecting serum responses against H. pylori in actively infected and eradicated individuals. Moreover, it allows stratifying patients according to their disease state.

Figure 1

recomLine test results from patients without and with eradication therapy. (a) recomLine positivity, (b) mean recomLine score, (c) immune response frequency, and (d) mean immune response intensity of individuals with an active ongoing H. pylori infection (n_{Hp positive} = 402) compared to individuals having received antibiotic treatment in the past (n_{years since eradication} = 210), indicated as years since eradication and grouped (0–5, 6–11, and 12–17 years) analysing a German cohort (DE). Moreover, the relationship between (e) immune response frequency to individual antigens (●CagA, ■VacA, ▲GroEL, ▼UreA, ♦HcpC, and ○gGT) and (f) percentage of isolated CagA immune response and the time since eradication therapy analysing a Dutch cohort (NL) (n = 65). While the immune response for all antigens tested declines after eradication, the response towards CagA antibodies persists over a long period, resulting in an isolated CagA positivity, which remains above cutoff and is significantly increased (p < 0.01) in the 12–17 years group compared to all other antigens.

Figure 2

Box plot diagrams showing the association between recomLine test results (dark grey: score recomLine/light grey: number of positive antigens) and histological findings in H. pylori positive cases. (a) Correlation between recomLine test results and the chronicity of inflammation (n = 391). (b) Correlation according to the activity of inflammation (n = 389) as well as (c) pathological findings (n = 95) according to histology. Data are presented as box plot showing the median, 75th and 25th percentile, outlier, and extreme values. Significances were calculated using the Kruskal-Wallis test. The results show a significant correlation between the achieved recomLine score and histological findings. The sum of positive antigens only correlates significantly with the chronicity of inflammation.

Figure 3

Association between positivity of individual antigens (●CagA, ■VacA, ▲GroEL, ▼UreA, ♦HcpC, and ○gGT) and histologic findings such as (a) chronicity and (b) activity of inflammation as well as (c) most severe lesions. The chronicity and activity are categorized as mild, moderate, and severe. Most severe lesions are categorized as either absent, atrophy, intestinal metaplasia, ulcer, or gastric cancer (GC) according to histology. A significant correlation could be found between CagA or VacA and the chronicity of inflammation (p = 0.015; p < 0.01, resp.), as well as CagA and the activity of inflammation (p < 0.01).

Figure 4

Association between the type of serological response (I/II) and the findings in histology analysing H. pylori positive cases. The presence of an immune response against H. pylori virulence factors CagA/VacA indicates a type I infection, and these are defined as the high-risk group. The chronicity and activity are categorized as mild, moderate, and severe. Most severe lesions are categorized as absent, atrophy, intestinal metaplasia, ulcer, and gastric cancer according to histology. The proportion of type I immune response increases with higher degrees of chronicity and activity of gastritis (p = 0.02 and p < 0.01, resp.). There is a significant increase in the portion of type I immune response if signs of advanced pathology are present in histology (p < 0.01).

Figure 5

H. pylori diagnostic decision tree. Low risk: no seropositivity against CagA and VacA and no additional risk factors; +Risk factors: no seropositivity against CagA and VacA but additional risk factors such as smoking, diet, and genetic predisposition; High risk: seropositivity against CagA and VacA.