LACK OF ASSOCIATION BETWEEN HELICOBACTER PYLORI'S VIRULENCE AND INCREASED SERUM C-REACTIVE PROTEIN LEVELS IN FUNCTIONAL DYSPEPTIC PATIENTS

ABSTRACT

Background

Recently, a great variety of studies aimed to investigate and even suggestHelicobacter pylori as an important key factor in gastrointestinal and non-gastrointestinal events development. The well-established relationship between bacterial virulence and increased risk for peptic ulcer or gastric carcinoma is not so clear when comparing inflammation markers alterations, such C-reactive protein, with the pathogen.

Objective

The objective of this study was to evaluate the presence of H. pylori, bacterial virulence and C-reactive protein serum levels in individuals diagnosed with functional dyspepsia.

Methods

Were prospectively included in this study 489 dyspeptic individuals. They fulfill Rome III clinical criteria for the diagnosis of functional dyspepsia with no organic disease at endoscopy. The bacterial infection was established by histology and urease rapid test. The levels of serum C-reactive protein were obtained by immunonefelometry and CagA status ofH. pylori positive individuals was determined through an imunoenzimatic assay.

Results

Prevalence rate of H. pylori was 66.3% and virulence factor CagA was detected in nearly 43% of positive samples. In addition, it has been noticed an association between Ilex paraguariensis(yerba maté) consumption and pathogen's prevalence. An important effect of bacterial infection on inflammation was only observed in gastric epithelium.

Conclusion

No systemic response to the pathogen, measured through C-reactive protein levels, was observed, regardless of CagA status. Otherwise, the intake of yerba maté should be considered as a cultural factor possibly related toH. pylori's transmission.

HEADINGS
Helicobacter pylori; Dyspepsia; C-reactive protein; Virulence factors; Inflammation

RESUMO

Contexto

Recentemente, uma grande variedade de estudos tem investigado e até mesmo sugerido a presença de Helicobacter pylori como um importante fator no desenvolvimento de eventos restritos ou não ao trato gastrointestinal. A relação já bem estabelecida entre virulência bacteriana e risco aumentado para úlcera péptica ou adenocarcinoma gástrico não parece estar tão elucidada quando se comparam alterações de marcadores inflamatórios, como a proteína C-reativa, com a presença do patógeno.

Objetivo

O objetivo deste estudo foi avaliar a presença da infecção por H. pylori, a virulência bacteriana e os níveis séricos de proteína C-reativa em indivíduos diagnosticados com dispepsia funcional.

Métodos

Foram incluídos neste estudo, prospectivamente, 489 indivíduos dispépticos. Os pacientes deveriam preencher os critérios clínicos de Roma III para o diagnóstico de dispepsia funcional sem apresentar doença orgânica evidenciada a partir da endoscopia. A infecção bacteriana foi estabelecida por histologia e pelo teste rápido da urease. Os níveis de proteína C-reativa foram quantificados através de imunonefelometria e o status para a presença da CagA dos indivíduos infectados por H. pylorifoi determinado por ensaio imunoenzimático.

Resultados

A taxa de prevalência de H. pylori foi de 66.3% e o fator de virulência CagA foi detectado em aproximandamente 43% das amostras positivas. Adicionalmente, denotou-se uma associação entre o consumo deIlex paraguariensis (chimarrão) e a prevalência do patógeno. Um importante efeito da infecção bacteriana na inflamação apenas foi observado localmente, no epitélio gástrico.

Conclusão

Não foi evidenciada resposta sistêmica ao patógeno aferido através dos níveis de proteína C-reativa, independentemente do status para CagA. Por outro lado, o consumo de chimarrão pode ser sugerido como um fator cultural possivelmente relacionado à transmissão de H. pylori.

DESCRITORES
Helicobacter pylori; Dispepsia; Proteína C-reativa; Fatores de virulência; Inflamação

INTRODUCTION

Since Helicobacter pylori (H. pylori) was described by Robin Warren and Barry Marshal, in 1982, this bacterium opened a new period in the gastric microbiology diagnosis and therapeutics8 24. H. pylori causes one of the most prevalent infections in human beings with a worldwide distribution that can reach relative frequencies that vary from 20% to 90% in different populations33 35. The microorganism exhibits a high tropism to the gastric epithelium, where can causes immune and inflammatory responses that may persist for all life if not eradicated5 10.

This pathogen, that initially was included in the Campylobactergenus, is a Gram-negative curved bacillus, presenting 2-6 flagella and an ability to produce urease abundantly16 24. Other virulence factors such CagA protein have been studied extensively7 23 40. According to the recent reports, CagA positive strains can cause severe damage to the gastric epithelium, being related specially with increased levels of interleukin-8 (IL-8), gastroduodenal ulcers and gastric neoplasia occurrence26 29 41. In addition, this virulence factor has been associated with systemic inflammation, resulting in high serum C-reactive protein (CRP) levels and the bacteria presence being related to higher cardiovascular risks21 37.

Some behaviors have been associated to the transmission of H. pylori. Studies have reported that several aspects can be related to incidence and prevalence rates like socioeconomic status, years of study, institutionalization practice or social habits8 20. In 2010, a Brazilian study has demonstrated an anti-H. pylori activity of plant extracts, like yerba maté tea (Ilex paraguariensis), but no association between bacteria prevalence and tea consumption has been previously described6.

All H. pylori's infected people present histological gastritis. Moreover, the bacteria, classified as type I carcinogen32, has been associated with the pathogenesis of gastric and duodenal peptic ulcers, with gastric carcinoma and with the gastric MALT lymphoma11. Some studies also suggest a bacterial role in cardiovascular disorders, referring to the inflammatory process that results in the atheroma formation and evolution1 17. Additionally, one of the most challenging questions related to H. pylori is the bacterium association with the functional dyspepsia12 25 27.

As the influence of H. pylori CagA positive strains and potential changes in systemic inflammation remains a debatable matter. Thus, our study aimed to verify a possible relationship between bacterial virulence and systemic and/or local inflammation, through the measurement of CRP levels in serum and the comparison with the histological gastric mucosa analysis from functional dyspeptic patients.

METHODS

Patients

Between November 2006 and June 2008, 489 subjects who had undergone upper gastrointestinal endoscopic evaluation to participate in HEROES trial (Helicobacter Erradication Relief of Dyspeptic Symptoms), at Hospital de Clínicas de Porto Alegre, Porto Alegre, state of Rio Grande do Sul, Brazil, were prospectively and consecutively included in this study.

As inclusion criteria, the patient should meet the Rome III criteria which includes at least one of the following symptoms: a. bothersome postprandial fullness; b. early satiation; c. epigastric pain; or d. epigastric burning. Individuals presenting organic disorders diagnosed by upper gastrointestinal endoscopy, such as esophagitis, gastric or duodenal peptic ulcers, neoplasia or other conditions that could be the cause of the referred symptoms were excluded from the study. Conditions such previous treatment to H. pylori, clinical manifestations of organic diseases or presence of significant comorbidities and non-acceptance of the intervention by the patient were equally considered exclusion criteria.

Additionaly, after informed consent form assignment, subjects answered questions about medical history, dietary habits and quality of life.

Endoscopic procedures

It was performed with a videoendoscope (GIF-100, Olympus Co). After 8 hour fast, patients were sedated intravenously according to their age, weight and tolerance to fentanyl or meperidine plus midazolam. From each patient were collected three antral fragments, one specimen from the gastric body, and one from theincisura angularis region. Biopsy samples were submitted to a H. pylori investigation by two distinct methodologies: histology and rapid urease test.

Helicobacter pylori diagnosis

A biopsy specimen from each gastric region (antrum, body and incisura angularis) was placed in a 0.5 mL Christensen solution (Uretest^(r), Renylab) to verify the presence of bacterial urease. A positive result was reported if there was a color change from yellow to pink within 12h of incubation at room temperature according to the manufacturer's instructions.

Concurrently, paired biopsy specimens were collected from antrum, fixed with 10% formol and stained for Hematoxylin and Eosin (H&E) and Giemsa. Two independent, experienced and blinded pathologists performed the histological examination, and discordances were solved by a third expert's opinion. The inflammatory status was determined according to Sydney's Endoscopic Classification, as previously described14. H. pylori's infection was considered present according to the positivity in both methodologies (rapid urease test and histological evaluation).

Determination of CRP levels

Approximately 10 mL of whole blood were collected from each patient and after 3000 rpm centrifugation during 15 minutes, serum samples were stored at −80°C and analyzed for high sensivity C-reactive protein (hsCRP) and anti-CagAH. pylori antibodies, as described below.

CRP levels were measured by a immunonephelometric assay (CardioPhase^(r) hsCRP Dade Behring) on a Behring Nephelometer II analyzer. The detection limit for CRP was 0.17 mg/L, and the measuring range was 0.175-1100 mg/L.

Determination of cagA status

Serological search for anti-CagA was performed with a commercial available kit (CagA IgG EIA WELL^(r), Radim) according to manufacturer's instructions. After incubation, plates were read in a spectrophotometer at 450 nm and samples with IgG values higher than 15 RU/mL were considered reactive for anti-CagA IgG antibodies. Each sample was measured twice to ensure the precision of the method.

Ethical considerations

All study procedures were conducted in agreement with Declaration of Helsinki, with the Brazilian Federal Resolution 196/96 and were approved by our local institutional review board, inscribed as project number 07-547. All patients were informed about the study's objectives and subsequently provided written informed consent before any intervention.

Statistical analysis

Data are presented as mean (SD) or median (25th - 75th percentile), when otherwise stated. Quantitative variables were first analyzed concerning Gaussian distribution and assessed with t-test and one-way ANOVA (parametric) or Mann-Whitney and Kruskal-Wallis test (non-parametric). Categorical variables were described by absolute and relative frequencies and analyzed using chi-square test with adjusted residuals test. The analysis was performed using SPSS v. 18. A P value was considered significant if <0.05.

RESULTS

The characteristics of the subjects are shown in the Table 1. Regarding to the gastric H. pylori status, 66.3% of the functional dyspeptic patients were H. pylori-positive. Among these patients, 42.8% presented antibodies against CagA virulence protein.

TABLE 1
Sociodemographic characteristics and life style of study population according to H. pylori status and virulence

• SD: standard deviation; n: number of subjects; ^a Student's t-test used for continuous variables e Chi-square test used for categorical variables; ^b Missing data not considered (Student's t-test used for continuous variables and Chi-square test used for categorical variables); ^c Considering missing data (ANOVA one-way used to continuous variables and Chi-square test used for categorical variables)

Relevant findings include i. a higher prevalence of H. pylori in the population with less than nine years of education and ii. no association between bacterial frequency and gender, race, smoking habit or alcohol consumption was observed.

It was also asked to the patients about the habit of drinking yerba maté tea, and 45.3% of positive H. pylori individuals reported regular consumption of such beverage (P=0.006).

Table 2 shows local and systemic inflammatory status according to the presence of H. pylori and CagA virulence factor. In general, it was observed a high association between the presence of anti-CagA and a high inflammation and inflammatory activity in the gastric epithelium (P<0.001), without significantly affect the CRP values. Regarding the systemic inflammation marker (hsCRP) and local inflammatory activity or inflammation grade in dyspeptic patients, no association was observed between the systemic inflammation marker and an inflammatory activity (P=0.339) nor between systemic inflammation marker and inflammation grade (P=0.508).

TABLE 2
Local and systemic inflammatory status according to the presence ofH. pylori and cagA virulence factor

• ^a
  Kruskal-wallis test used for continuous variables and Chi-square test used for categorical variables; ^b Statistically significant association by adjusted residuals test 5% of significance.
• hsCRP: high sensivity C-reactive protein.

Discussion

Recently, H. pylori and has been suggested as an important factor in extra-gastric manifestations such as increased serum CRP levels and high systemic inflammation leading to a higher potential risk factor to the development of cardiovascular diseases17 28. This study was developed in order to verify a possible relation between bacterial virulence and systemic and/or local inflammation through the measurement of CRP levels in serum and the histological evaluation of gastric mucosa of functional dyspeptic patients.

Regarding the bacterial influence on the immune system, a study conducted by Lee et al. (2010) indicates that H. pylori infection or their lipopolysaccharide stimulation led to significant increased expressions of inflammatory mediators including tumor necrosis factor-alpha (TNF-α), IL-8, inducible nitric oxide synthase and cyclooxygenase- 222. The mechanism of this response affects the innate immunity through the recognition of some conserved m crobial constituents by receptors expressed on host-epith lial cells as well as neutrophils. In the gut, such recognition results in the activation of conserved signaling cascades mediated by nuclear factor kB (NF-kB), mitogen-act vated protein kinases and caspase-dependent signaling pathways30.

The main findings of our study were: 1. no association between CRP serum levels inH. pylori infected patients with functional dyspepsia, independently of CagA status; 2. a remarkable inflammation and inflammatory activity in gastric epithelium of patients carrying the most virulent strain; and 3. a high prevalence of H. pylori in patients with less than nine years of education; and, 4. in those who mentioned yerba maté tea consumption.

Regarding the first finding, our results do not agree with some reports that have shown a considerable association of H. pylori and increased serum CRP levels, especially in positive-CagA strains18. Considering the possible association between higher systemic inflammation levels and H. pylori infection, some data show that after the pathogen eradication, serum CRP levels can decrease significantly2 19. Otherwise, there is no consensus about this matter. For example, a strong study conducted by Brenner and cols (1999) involving more than 1.800 healthy subjects did not prove the relation between H. pylori, bacterial virulence and inflammation markers. Although it was observed an inverse relation betweenH. pylori infection and serum albumin, the bacteria presence was unrelated to C-reactive protein and the leukocyte count, regardless of CagA status4. Such result was also reported in several studies that showed no impact of H. pylori's infection in systemic markers of inflammation13 15 36 38.

CagA is the most extensively investigated virulence factor of H. pylori being encoded by cytotoxin-associated genes pathogenicity island (cagPAI)3. A strain expressing cagA protein, which is present in more virulent isolates, is typically associated with the production of proinflammatory cytokines, especially IL-8, and such factor has been reported as an important factor related to the peptic ulcer and gastric adenocarcinoma occurrence41. It was observed a remarkable inflammatory activity and inflammation in the gastric epithelium of those people with the most virulent strain, whereas, neither inflammatory activity nor inflammation were observed in more than 90% and 54% of H. pylori's negative subjects, respectively.

We also found an important association between prevalence of the pathogen and years of education and yerba maté tea consumption. As previously appointed by other Brazilian researches, 66% of our study population presented H. pylori's infection, among these patients, nearly 58% reported less than 9 years of study. This finding has been also observed in other studies and possibly indicates socioeconomic status and years of study as important conditions associated to the risk factors for H. pylori's transmission9 33.

According to data previously reported (Kodaira et al.), our findings support thatH. pylori frequency is not related with smoking habit or alcohol consumption20. On the other hand, we report here, for the first time, the association of H. pylori's infection with the cultural habit of drinking maté. Maté is an infusion of the herbIlex paraguariensis that is prepared in a gourd and is drunk very hot through a metal straw. The infusion is shared by different people using the same gourd and straw. Although it has been reported that the herb presents anti-H. pylory activity6, this cultural habit may provide a possible route for the bacterial transmission.

Considering our study procedures, we recognize that CRP is an unspecific marker of acute inflammation and that it can be related to a several diseases and conditions. Firstly described by Tillett e Francis (1930), this inflammatory protein has an important role to predict the cardiovascular risk, among other applications31 39. Although we have evaluated systemic inflammation through this analyte, we agree that other parameters should be useful and more specific to determine a possible link between H. pylori's infection and systemic inflammation. In example, some studies have considered to study as inflammation markers interleukin-6 (IL-6), IL-8 and TNF-α, which seems to be specially related to bacterial infection3 34.

As mentioned above, regarding the remarkable association between the presence of anti-CagA antibodies and higher tissue damage, although with no systemic responses through the measurement of hsCRP levels, we encourage the development of further studies involving additional inflammatory markers that could be related to higher systemic inflammation in functional dyspeptic people carrying H. pylori and its virulence factor.

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  Disclosure of funding: no funding received

Publication Dates

• Publication in this collection
  Jan-Mar 2016

History

• Received
  17 June 2015
• Accepted
  12 Sept 2015