Peer Review #2 of "The increasing prevalence of HIV/Helicobacter pylori co-infection over time, along with the evolution of antiretroviral therapy (ART) (v0.1)"

Helicobacter pylori (H. pylori) is one of the most common human bacterial infections with prevalence rates between 10-80% depending upon geographical location, age and socioeconomic status. H. pylori is commonly found in patients complaining of dyspepsia and is a common cause of gastritis. During the course of their infection, people living with HIV (PLHIV) often have a variety of gastrointestinal symptoms including dyspepsia and while previous studies have reported HIV and H. pylori co-infection, there has been little data clarifying the factors influencing this. The aim of this case-control study was to document the prevalence of H. pylori co-infection within the HIV community as well as to describe endoscopic findings, gastritis topography and histology, along with patient demographic characteristics across three different periods of time during which antiretroviral therapy (ART) has evolved, from prehighly active antiretroviral therapy (HAART) to early and modern HAART eras. These data were compared to well-matched HIV negative controls. Two hundred and twelve PLHIV were compared with 1617 controls who underwent their first esophagogastroduodenoscopy (EGD) to investigate dyspepsia. The prevalence of H. pylori co-infection among PLHIV was significantly higher in the early (30.2%) and modern HAART period (34.4%) compared with those with co-infection from the pre-HAART period (18.2%). The higher rates seen in patients from the HAART eras were similar to those observed among HIV negative controls (38.5%). This prevalence increase among co-infected patients was in contrast to the fall in prevalence observed among controls, from 60.7% in the early period, to 52.9% in the second observed period. The three PLHIV co-infected subgroups differed regarding gastritis topography, morphology and pathology.

H. pylori has the main role in the pathogenesis of chronic gastritis, peptic ulcer disease, gastric cancer, MALT lymphoma and several extra-gastric manifestations (5, 6). It is one of the most common bacterial pathogens affecting the general population (7) and its prevalence is estimated to be up to 50% worldwide (8). The risk factors and routes of transmission still remain unclear. The most important risk factor seems to be a lower socioeconomic status in early childhood, allowing easy person-to-person transmission (9, 10).
The prevalence of H. pylori infection among PLHIV varies from 10 to 76%, depending on the time period, geographical location and population (4, [11][12][13][14]. Earlier studies reported lower prevalence of this co-infection among PLHIV compared to matched HIV negative controls, contrary to data from the recent HAART era, where the H. pylori prevalence rate in PLHIV approaches the rate observed in general population. The aim of this study was to analyze the prevalence H. pylori co-infections, within the HIV community as well as to describe endoscopic findings, gastritis topography and histology, along with patient demographic characteristics across three different periods of time, during which antiretroviral therapy (ART) has evolved, from pre-HAART (1993)(1994)(1995)(1996)(1997), to early HAART (1999)(2000)(2001)(2002)(2003) and the modern HAART era (2011)(2012)(2013)(2014)(2015).

MATERIAL AND METHODS
This retrospective case-control study was carried out at the Hospital for Infectious and Tropical Disease of the Clinical Center of Serbia at the Faculty of Medicine, University of Belgrade, which is our the major reference center for the management of HIV infections. The study included HIV positive patients who underwent esophagogastroduodenoscopy (EGD) due to dyspeptic symptoms.

Study population
For the purpose of this study, patients from the PLHIV cohort were stratified into three categories based on the time of EGD performance: The first group (G1) consisted of patients from the last five years of the pre-HAART period, who underwent EGD between January 1 st 1993 and December 31 st 1997 (the pre-HAART group). The second group (G2) included patients who underwent EGD between January 1 st 1999 and December 31 st 2003, the first five years of HAART (the early HAART group). The third group (G3) included patients who underwent EGD between January 1 st 2011 and December 31 st 2015 (the modern HAART group). As HAART became available in Serbia in 1998, patients who underwent EGD from January 1 st 1998 to December 31 st 1998 were excluded from study in order to preserve the homogeneity of the groups.
The H. pylori/HIV co-infection prevalence and demographic characteristics across three groups were compared with the same parameters of HIV-negative controls who underwent EGD during the same three periods of time. Controls were matched with the study population.
Each patient/control was included in one HAART period group only. The study inclusion criteria were: dyspepsia (including epigastric discomfort, nausea, vomiting, heartburn, burping, loss of body weight, bloating and stomach cramps) lasting up to three months before their first EGD (after informed consent), with gastric biopsy and histology.

The study inclusion and exclusion criteria
The study exclusion criteria were: gastrointestinal bleeding, odynophagia, dysphagia, chest pain, chronic diarrhea (which could have jeopardized results) and being under 18 years of age.  1, Figure 1). 7 The age and gender of patients, across all three groups did not differ significantly, although there were more males in G3 than in G2 (p=0.012). In all three groups the majority of patients had secondary education; the level of education did not differ between G1 and G2 (p=0.719) while G3 patients had somewhat higher level of education than G1 (p=0.007) and G2 (p=0.044). The median CD4 cell count/µL was significantly different across groups [97 count/µL (4-545), 250 count/µL  and 435 count/µL (2-1622), in groups G 1-3, respectively, p<0.001]. G3 patients had the highest CD4+ counts, while the lowest count was observed in G1 patients. A significantly lower prevalence of AIDS was observed in G3 than in G2 patients. The level of HIV viremia did not differ significantly between G2 and G3. The level of HIV viremia had not been measured in pre-HAART era (Table1).

Data collection and instruments
The median duration of HIV infection from initial diagnosis to the EGD was significantly longer in There were 30 (52.6%) intravenous drug users (IVDUs) in G1, while the sexual route of HIV transmission was more prevalent among patients in groups G2 and G3 (p<0.001). There was no significant difference in the route of HIV transmission between G2 and G3 (p=0.101) ( Table 1).
In the latter two groups the number of patients with pathological EGD findings was similar (Table   3).
In PLHIV, the gastritis topography significantly differed between H. pylori positive subgroups (p<0.001). EGD revealed antral gastritis as the most common in G1 H. pylori positive subgroups patients. The majority of G2 H. pylori positive subgroups patients had either antral or pan-gastritis, while G3 H. pylori positive subgroups patients mostly suffered from pan-gastritis. The histopathological grading of gastritis activity was similar between subgroups, while H. pylori Formatted: Font:Italic 8 density differed significantly (p<0.001). The most severe density was recorded in G3 H. pylori positive subgroups patients. Although gastritis activity was similar across subgroups, the mildest cases prevailed in G1, and in G3 there were more patients with moderate activity (p=0.026). The intensity of lymphoid follicles, mononuclear cell infiltration, and the intestinal metaplasia were similar across subgroups. All cases of gastritis in the presented H. pylori positive cohort were classified as chronic non-atrophic gastritis. Intestinal metaplasia was only sporadically present, mostly replacing one to few foveolae. (Table 3).
Patients in G1 group with H. pylori co-infection were younger than G2 and G3 subgroups (p=0.018). Gender distribution was similar across H. pylori positive subgroups. The median CD4 cell counts were lower in G1 and G2 than in G3 H. pylori positive subgroups patients (p<0.001) ( Table 4).
Age of controls differed significantly between three HAART periods. Control patients from the modern HAART were significantly older than control patients from the pre-HAART and early HAART periods (p<0.001) ( Table 5).
The prevalence of H. pylori infection significantly varied between control groups, decreasing over time from 60.7% in pre-HAART to 38.5% in modern HAART era controls. In modern HAART controls, the prevalence of H. pylori infection was similar to the prevalence of H. pylori infection co-infected modern HAART individuals (Figure 2).

DISCUSSION
From the very beginning of the HIV/AIDS epidemic in Serbia, it became evident that these patients suffered from various gastrointestinal symptoms, including dyspepsia. The cause of gastrointestinal symptoms could be HIV per se, opportunistic and non-opportunistic infections including Helicobacter pylori (H. pylori), along with adverse effects of highly active antiretroviral therapy (HAART). Therefore, EGD has become an important differential diagnostic tool in the evaluation of gastrointestinal disease in PLHIV (16, 17). ART naïve PLHIV live with the impairment of cell-mediated immunity and are therefore at high risk for opportunistic infections and opportunistic tumors. Introduction of ART in 1986, which evolved from rather a weak mono and/or dual therapy to more potent HAART in 1996, has dramatically changed the course of HIV 9 infection, converting in to chronic, not necessarily a fatal disease. Good patient adherence to treatment and infection monitoring are of crucial importance (18, 19).
Occasionally, however, especially among late presenters in which HAART is initiated only at a point of profound immunodeficiency, the immune system recovery may be accompanied with the emergency of complications called the immune reconstitution inflammatory syndrome (IRIS).
IRIS is a consequence of the restored immune response to specific infectious or non-infectious antigens (20-23). According to our data, the prevalence of HIV/ H. pylori co-infection in Serbia ranges from 18.2% to 34.4%, depending on the study period, namely the type of applied ART. To the best of our knowledge, this is the first EGD-based morphological comparison of HIV/H. pylori co-infected patients in three different periods of ART evolution. We have demonstrated a significantly reduced incidence of pathological EGD findings in PLHIV with dyspeptic symptoms in the modern HAART era in comparison with PLHIV co-infected during the pre-HAART era.
Then, in three time points H. pylori gastritis topography has also changed significantly between the three HAART periods. During the pre-HAART era, gastritis was characterized by mild H. pylori density and moderate activity. This is contrary to gastritis observed in the modern HAART group, where the density became mostly severe, while the activity remained mild. The changes in gastric histological findings may be associated with an increased CD4 cell count in H. pylori positive subgroup, and could suggest that the type and duration of immune and inflammatory responses are responsible for differences in histological presentations of H. pylori associated gastritis. The direct impact of ART on H. pylori infection and gastritis should also be taken into consideration, especially since there is evidence for ART-induced GI symptoms (27). In the modern HAART era, PLHIV are living longer, are normally integrated in society, with similar risks and diseases as HIV-negative individuals.

Study limitations
This study has several limitations. First, it is a retrospective case-control study. Second, various risk factors for H. pylori acquisition, such as exposure to antibiotics, as well as to proton-pump inhibitors (PPI) and H2 blocker therapies, along with hypochlorhydria, body mass index, particular habits, which have all been addressed by numerous studies before (12,13,25,26), were not explored. Third, we have not sufficiently explored H. pylori acquisition in HIV negative cohorts. Despite these limitations, we believe that the study represents new research findings on the relationship between 11 ART development over time, HIV risk factors, and H. pylori infection, gastritis distribution and topography.

CONCLUSION
Our data may also suggest that H. pylori needs a functional immune system to induce human gastric mucosa inflammation. It is necessary for the future studies to investigate the difference between epidemiological characteristics of H. pylori infection in PLHIV and HIV negative population, expanding conclusions and implications from our study. Altogether, our findings suggest that HAART has an important impact on the EGD features of HIV/H. pylori co-infection.