Seroprevalence of helicobacter pylori in human immunodeficiency virus-positive Patients and it's correlation with CD4(+) Lymphocyte Count.

BACKGROUND: This study assessed the seroprevalence of Helicobacter pylori antibodies among Iranian patients with human immunodeficiency virus (HIV) infection. It also examines whether anti H. pylori seroprevalence was associated with the severity of the HIV infection or the antiretroviral treatment.
MATERIAL AND METHODS: A total of 114 HIV-infected patients and 114 age and sex-matched controls, without symptoms referable to upper gastrointestinal tract were recruited. Blood samples were obtained from all subjects. Serum IgG and IgA against H. pylori measured using the enzyme-linked immunosorbent assay (ELISA).
RESULTS: The rate of anti H. pylori IgG seropositivity was 57.9% in HIV-infected patients and 28.95% in controls (P < 0.001), while the rate of IgA seropositivity was 2.64% in HIV patients and 31.57% in controls (P < 0.001). Although there was an increasing trend of higher IgG and IgA titre by increasing CD4 cell count in HIV-positive patients, it was not reach statistical significance. There was no statistical difference in the serology of anti H. pylori IgG and IgA between patients receiving antiretroviral therapy comparing untreated HIV patients.
CONCLUSIONS: This study showed higher seroprevalence of H. pylori IgG along with lower seroprevalence of H. pylori IgA in HIV-positive patients compared matched controls.

INTRODUCTION

Patients with human immunodeficiency virus (HIV) are susceptible to many different gastrointestinal (GI) opportunistic infections. HIV infection increase the colonisation of pathogens in GI tract.123

Helicobacter pylori, a gastric flagellate Gram-negative rod bacterium, is considered as the major aetiology of chronic gastritis and peptic ulcer disease.4 Over half of the worlds' population is estimated to be infected with H. pylori.567 The prevalence of H. pylori infection vary across different geographical regions with the range of 32% and 65%.89101112 The infection has been seen in more than 90% of the patients with gastritis131415 and 70% to 100% of those with peptic ulcer diseases.61011 H. pylori is also known to have carcinogenic effects16 , and National Institute of Health Consensus Development Conference Statement recommends to eradicate this bacterium (if confirmed) in any cases of peptic ulcer.17

The overall prevalence of H. pylori is suggested to be correlated with socioeconomic conditions. Gender, occupation, low socioeconomic status, educational level, and alcohol consumption are known risk factors for H. pylori infection.15 However; persistent colonisation depends on the host immune responses to the bacterium.

Although some studies have shown that H. pylori infection is less common among HIV-positive individuals with GI symptoms121819202122 , other investigations232425 suggested a higher prevalence of H. pylori infection in HIV-positive patients as a result of immune suppression. Hence, the relationship between H. pylori infection and HIV remain controversial.2627

Moreover, there are limited data regarding the seroprevalence of H. pylori in HIV-positive patients, particularly in our region. This study aims to assess the seroprevalence of H. pylori infection among HIV-positive patients and its correlation with CD4+ cell count and some of hematological parameters. It also examines whether H. pylori seroprevalence is related with severity of HIV infection, or advanced stage of the disease and the administered antiretroviral regimens.

MATERIALS AND METHODS

A case- control study was carried out at the center of high-risk behavioural disease in Imam Hospital Complex, a major referral hospital in Tehran, capital of Iran, affiliated to Tehran University of Medical Sciences (TUMS). One-hundred and fourteen patients already diagnosed as HIV-positive cases attended our center, between January 2010 and June 2011, were consecutively enrolled in this study. Controls were subjects with negative test result for HIV, and recruited from the same centre. Controls were individually matched to cases with respect to sex and age (± 2 years) and where possible socio-economic status. None of the HIV-positive patients and controls had symptoms referable to upper GI tract. Subjects with a history of autoimmune diseases, malignancies such as lymphoma, peptic ulcer disease, documented diagnosis of viral infections within the past month, those who had received corticosteroid, antibiotics within the past 4 weeks, and subjects were previously treated for H. pylori infection were excluded.

The study was approved by Research Ethic Committee of TUMS and informed consent was obtained from each patient before enrollment in this study. Diagnosis of HIV infection confirmed with serology, polymerase chain reaction (PCR) or Western blot following the recommendation of National AIDS Control Organization (NACO 2007).

Five millilitres clotted blood and 3 cc anticoagulated blood with EDTA were obtained from subjects. The clotted blood was centrifuged in 3000 g for 15 minutes. Extracted serum was then stored in a -70 centigrade Celsius freezer. Ig A and Ig G anti-H. pylori antibody titretitre was measured by ELISA techniques in room temperature using Mono bind Inc, Lake Forest, CA, USA kit. Following the instruction provided by the manufacturer, values upper than 20 μ/ml were considered positive. Anticoagulated blood was also assessed in terms of CD4+ and CD8+ lymphocytes cell count by flow cytometry device (FCM) (PARTEC, Japan).

Determination of AIDS status was done according to the guidelines of World Health Organization (WHO). HIV patients with CD4 count below 200 cells/ml or specific clinical conditions suggestive of advanced immunodeficiency infection were categorised as AIDS stage.

Data were analyzed using Statistical Package for Social Sciences (SPSS version 18, Chicago, Inc). Data were presented as mean ± standard deviation (SD). Anti-H. pylori Immunoglobulin titres were expressed as Median and interquartile ranges (25th -75th centile). Chi-square test was employed to compare the seroprevalence of H. pylori infection between groups. Students't test and one-way ANOVA test were employed to declare the trend in each parameter between different groups. The distribution of anti-H. pylori IgG was shown using boxplot graph. P-value less than 0.05 was considered statistically significant.

RESULTS

A total of 114 HIV-positive patients and 114 controls were included in this study. Values of anti-H. pylori IgG and IgA anti bodies higher than the cut-off level of 20 u/ml were considered seropositive. The demographics and seroprevalence of anti-H. pylori Ig subclasses among HIV patients and controls were shown in Table 1. There were no significant difference regarding the age, gender, residence, and educational status between HIV patients and controls.

Table 1

Demographic characteristics and seroprevalence of anti-H. pylori Immunoglobulin subclasses HIV patients and controls

IgG antibody titre was positive in 66 HIV-positive patients (57.9%) which revealed a statistically significant difference (P<0.0001) when compared with 33 HIV-negative patients (28.95%) [Table 1]. On the other hand, IgA titre was positive in 3 HIV-positive patients (2.64%) compared to 36 HIV-negative patients (31.57%) which revealed a significant difference statistically (P<0.0001).

In HIV-positive patients, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and IgG titre had a significant relationship with H. pylori seropositivity (P<0.05). In contrast, other haematological parameters did not reveal a statistically significant difference [Table 2].

Table 2

Relationship of H. pylori seropositivity with CD4+ cell count, hematological parameters and some other biochemistry in HIV-positive patients

None of the HIV-positive patients showed clinical symptoms suggestive of advanced immunodeficiency infection. Hence, staging of AIDS was done according to the values of CD4+ count <200 cells/μl. Among 114 HIV patients, 37 were classified in AIDS stage. Figure 1 represents the distribution of serum anti-H. pylori IgG in AIDS, non-AIDS HIV patients and controls.

Figure 1

Distribution of serum anti-H. pylori IgG in AIDS, non-AIDS HIV patients, and controls. The boxplot shows the median, interqaurtile range, minimum, maximum and outliers of serum anti-H. pylori IgG

Of 114 HIV-positive patients, 79 (69.3%) were receiving antiretroviral therapy (ART). There were no statistical differences in the positive serology of anti-H. pylori IgG and IgA between patients undergone ART comparing untreated HIV patients (54.4% vs. 65.7%, P = 0.26 and 1.3% vs. 5.7%, P = 0.17).

To examine whether the severity of the HIV infection was associated with anti-H. pylori seroprevalence, HIV patients were classified into three groups based on their CD4+ cell count accordingly; CD4+ < 200, 200 ≥ CD4+ <500, and CD4+ ≥500. Serum anti-H. pylori IgG and IgA seroprevalence were compared between these groups [Table 3]. Although there was an increasing trend of higher IgG and IgA titre by increasing CD4 cell count in HIV-positive patients, it was not reach statistical significance.

Table 3

Anti-H. pylori seropositivity according different categories of CD4 cell count in HIV-positive patients

DISCUSSION

HIV infection is associated with different GI opportunistic infections, including cytomegalovirus (CMV), cryptosporidium, microsporidia and fungal oesophagitis.13 Whether HIV/AIDS infection has an impact on altering H. pylori prevalence remained controversial. Prevalence of H. pylori in HIV-positive patients has been reported in several studies181921222528293031 , either in eastern world with higher prevalence222829 of H. pylori infection or in the tropical countries with lower prevalence.232425 This variation would apparently be due to the differences in sanitation, educational level, age, life styles, and socioeconomic status of different cultures which have been shown to be of major impacts in colonisation of H. pylori and subsequent infection.3233

Present study found higher seroprevalence of anti-H. pylori IgG in HIV patients compared to controls. The presence of anti-H. pylori-specific IgG is considered as a marker of chronic infection with this pathogen. In this study, we tried to match the cases and controls regarding the gender, age, residence and educational level status to minimise the effect of socioeconomic status on our results. This study also categorised HIV patients into AIDS and non-AIDS stages based on the CD4 cell count, and found no significant difference in serum anti-H. pylori IgG distribution between two groups.

In contrast, Moges et al.28 was reported lower prevalence of H. pylori infection (19.6%) in Ethiopian HIV-positive population compared to HIV-negative dyspeptic patients (80.4%).

Studies by Hong-bin et al. and Lv et al were34 reported a strong relationship between H. pylori infection and the stage of HIV from asymptomatic to the AIDS stage and indicated that the lower prevalence of H. pylori infection in HIV-positive patients is due to the suppressed immune response. They used upper endoscopy and gastric mucosa biopsy to confirm H. pylori infection. This is of great importance to remember that H. pylori infection is confirmed by endoscopic studies and urease breathe test. Fialho et al.30 was evaluated the H. pylori status of HIV patients with dyspepsia by urease test and histology. They demonstrate lower prevalence of H. pylori in these patients compared with symptomatic controls. As the symptoms of dyspepsia are more common among HIV-positive population, taking medications such as PPIs and more attempts to eradicate H. pylori infection by the physicians may result in decreased infection rate of such microorganism.29 Decreased secretion of gastric acids has been accounted as another explanation34 ; hence, other opportunistic infections such as CMV may emerge to compete with H. pylori. This in addition to the decreased acid secretion may lead to inappropriate environment for colonisation of H. pylori.22232425353637 Other studies proposed a different role of H. pylori in peptic ulcerogenesis, and chronic active gastritis in HIV-positive patients.29

Interestingly, in this study there was a significant difference in the seroprevalence of different subclasses of Ig between HIV-positive and negative patients. HIV-positive patients had a higher rate of IgG seropositivity and a lower rate of IgA seropositivity compared to controls. It seemed like HIV infection had increased the level of IgG titre while it had decreased the level of IgA titre. Decreased gastric colonisation of H. pylori may be an explanation for this.30 However, it is not clear why level of IgG titre does not decrease in proportion to the decreased IgA level. Dysregulation of humoral and cellular immunity in HIV-positive patients may be a possible explanation for this pattern.38 While alteration in activity of humoral response may lead to a decrease in secretion of anti-H. pylori antibodies such as IgA, abnormal production of nonspecific polyclonal antibodies may explain the increased level of anti-H. pylori IgG antibodies. Previous studies have indicated that HIV infection by compromising the status of cellular immunity may result in decreased serum level of antibodies. However, this finding has been only shown in one study and in IgG subclass.28 Although it seems rational that HIV infection suppresses the ability of antibody production, our results demonstrated a different pattern. In contrast to the previous studies reporting lower seroprevalence of anti-H. pylori IgG, our study showed that IgG titre is higher in HIV-positive patients while the level of serum IgA is lower. It has been stated that serum levels of IgG and IgA are sensitive tests for H. pylori infection.39 Although it has been suggested that the positive cut off should be adjusted with age, this factor does not account in our results as controls were matched with cases regarding sex and age.

Haematological parameters were also compared between H. pylori seropositive and seronegative cases in HIV-positive group. Except MCV, MCH, no significant differences in other parameters were observed between H. pylori seronegative and seropositive HIV patients. There was no single study in the literature to be compared with this finding.

In the present study, an increase in seroprevalence of H. pylori is observed by increasing the CD4+ cell count which did not reach statistical significance. In contrast, Some investigations have shown a significant relationship between CD4+ cell count and H. pylori infection.28293134 This could be explained due to the excessive administration of antibiotics in HIV-positive patients to control the infectious complications.31

The diagnosis of H. pylori infection in our study was not possible due to lack of endoscopic studies and other gold standard methods. Other studies have performed endoscopic biopsy or urease breath test to confirm the diagnosis of this infection.91525293031 This is one of the limitations of our study as Fabris et al. have strongly alarmed about interpretation of anti-H. pylori IgG titre in HIV-positive patients.40

Future studies should evaluate the prevalence of H. pylori in HIV-positive patients using endoscopic studies and with special attention to the stages of HIV, administration of anti retroviral regimens and histological findings.

CONCLUSIONS

This study showed higher seroprevalence of H. pylori IgG along with lower seroprevalence of H. pylori IgA in HIV-positive patients compared to HIV-negative subjects.