1. Introduction
This study aims to explore the role of inflammatory biomarkers in predicting gastrointestinal side effects in HIV-positive patients undergoing modern antiretroviral therapy, with a focus on the impact of type II diabetes. By examining the levels of critical cytokines and markers of inflammation, this research seeks to identify predictors of GI discomfort in ART patients, particularly those with diabetes. Understanding these associations may help clinicians mitigate the side effects of therapy, improve treatment adherence, and ultimately enhance the quality of life for individuals living with both HIV and type II diabetes.
2. Materials and Methods
2.1. Study Design and Population
This study was designed as a cross-sectional, observational analysis to explore the association between inflammatory biomarkers and gastrointestinal side effects in HIV-positive individuals undergoing ART. The study population was divided into three distinct groups. The first group (120 patients), serving as the control group, included individuals who were HIV-negative and diabetes-free, providing baseline data for comparison on inflammatory biomarkers and gastrointestinal health. The second group (120 patients) consisted of HIV-positive patients undergoing antiretroviral therapy (ART) without type II diabetes, allowing for the analysis of ART-related inflammatory responses and gastrointestinal side effects in the absence of diabetes. The third group (80 patients) included HIV-positive patients who also had diabetes and were undergoing ART, facilitating the assessment of how type II diabetes influences both inflammation and gastrointestinal complications compared to the other two groups. We chose to focus solely on type II diabetes for this study because it is significantly more prevalent in the general population of HIV-positive individuals, and its association with chronic low-grade inflammation provides a more relevant context for examining the interaction between diabetes, antiretroviral therapy, and gastrointestinal side effects.
In this study, HIV patients were exclusively treated with standardized regimens of new-generation ART to reduce variability among participants and minimize drug-related toxic effects. Most patients received regimens based on integrase strand transfer inhibitors (INSTIs), such as dolutegravir, combined with nucleoside reverse transcriptase inhibitors (NRTIs), such as tenofovir alafenamide (TAF) and emtricitabine. These regimens were chosen for their excellent safety profiles and high efficacy, allowing us to minimize the confounding effects of drug-specific toxicities on gastrointestinal symptoms and inflammatory markers. All patients diagnosed with HIV were in their seventh month (after completing six months) of ART at the time their gastrointestinal symptoms were assessed, and inflammatory markers were re-measured. The period of six complete months was chosen as it represents a critical stage where the effects of ART typically stabilize, including viral suppression and partial immune recovery, minimizing variability from initial immune responses. This uniform timeframe ensured consistent exposure to ART across all participants, enabling more accurate comparisons between groups. Additionally, six months is an appropriate period to observe changes in inflammatory markers while still capturing ongoing inflammation influenced by factors such as type II diabetes.
For patients with type II diabetes, the therapeutic approach included antidiabetic medications with a low risk of gastrointestinal side effects, avoiding the use of metformin due to its potential to exacerbate gastrointestinal symptoms. Instead, medications such as SGLT-2 inhibitors (e.g., empagliflozin) and DPP-4 inhibitors (e.g., sitagliptin) were frequently used due to their favorable safety profiles. SGLT-2 inhibitors also provide additional benefits, including reduced cardiovascular risk and renal protection, without significantly affecting the gastrointestinal tract. Patients included in the ART + diabetes group had a preexisting diagnosis of type II diabetes prior to initiating antiretroviral therapy (ART). This approach ensured the evaluation of systemic inflammation and gastrointestinal symptoms in the context of established diabetes, eliminating the variability associated with diabetes induced by ART. By focusing on patients with diabetes diagnosed before ART, this study aimed to specifically analyze the interaction between this comorbidity and ART-related effects.
Both ART and antidiabetic regimens were meticulously documented and included in the statistical analyses to account for their potential influence on inflammatory markers and gastrointestinal symptoms. This standardized approach ensures a more precise assessment of the relationship between systemic inflammation, ART, and associated comorbidities while minimizing the confounding effects of medication-related toxicities.
The severity of gastrointestinal symptoms, including nausea, diarrhea, abdominal pain, and bloating, was assessed and classified using the Common Terminology Criteria for Adverse Events (CTCAE, version 5.0). According to this framework, symptoms were graded as follows: Grade 1 (mild), indicating minimal impact on daily activities; Grade 2 (moderate), defined as persistent or recurrent symptoms that interfere with daily activities and may require symptomatic intervention; and Grade 3 (severe), characterized by significant disruption of daily life and necessitating active medical management. For this study, clinical evaluations corroborated patient-reported outcomes to ensure consistency and objectivity in symptom grading. The majority of gastrointestinal symptoms were classified as Grade 2, with a subset of patients, particularly those with coexisting diabetes mellitus, exhibiting Grade 3 severity. This grading system allowed for a standardized and reproducible assessment of symptom severity across all study groups, providing a robust framework for evaluating the interplay between antiretroviral therapy, diabetes, and gastrointestinal side effects.
Demographic and clinical factors, such as age, gender, body mass index (BMI), and comorbidities, were considered across all groups to ensure robust and accurate comparisons.
Participants were selected from a large urban health center specializing in HIV care and management. The study was conducted at the “Dr. Victor Babeș Clinical Hospital for Infectious Diseases and Pneumophthisiology” in Timișoara, Romania. Two hundred forty people were enrolled in the study, with 80 HIV-positive patients who also had diabetes (ART + diabetes group), 120 patients in an ART group, and 120 in a control group. Biological data were collected at the initiation of treatment and again six months later, with patients being enrolled over a 10-year period from January 2013 to January 2023.
2.2. Inclusion and Exclusion Criteria
To ensure the study’s relevance and rigor, strict inclusion criteria were established for the patients involved in the research. These criteria aim to select patients with suitable profiles for assessing this study.
Inclusion criteria:
Participants with a confirmed diagnosis of HIV infection and type II diabetes.
Patients 18 years of age or older.
Patients in the ART group must be on antiretroviral therapy for six months.
Omnivorous patients who consume meat 5 to 7 days per week, given that dietary habits, including meat intake, can significantly influence inflammatory processes within the body.
Patients who agree to participate in the study and agree to sign the informed consent.
Participants who can comply with study requirements, including reporting to follow-up visits and collection of biological samples.
Clinically stable participants without signs of acute infections or other conditions that require urgent treatment and interfere with the state of health.
Patients have not been under treatment with immunosuppressants or anti-inflammatory drugs in the last year, as they could influence the level of inflammatory biomarkers.
Participants without known allergies to the substances that make up the ART treatments.
Participants must have the ability to understand and communicate effectively in Romanian to complete questionnaires accurately, provide reliable information during interviews, and fully comply with study requirements.
Exclusion criteria:
Patients with diagnosed autoimmune diseases such as systemic lupus erythematosus or multiple sclerosis are excluded because of the potential impact on inflammation and immune response.
Individuals who have reported a significant change in diet in the past 3 months.
Patients who have been recently diagnosed with severe non-HIV infections such as tuberculosis or severe pneumonia.
Patients with specific comorbidities that potentially impact gastrointestinal symptoms may be excluded from the study, as these conditions could confound the results and complicate the interpretation of the findings.
Patients who have used long-term corticosteroids for other conditions, as these drugs can suppress the inflammatory response.
Patients with kidney diseases that require specialized treatment or dialysis.
Patients with significant liver damage could complicate the interpretation of inflammatory markers.
Patients with cardiovascular diseases.
Patients who are currently undergoing treatment for cancer, including chemotherapy or radiotherapy, due to the potential impact on the immune system.
Patients who are known to be actively using illicit drugs or excessive alcohol are excluded, as these behaviors may affect study results.
Patients who have had severe adverse reactions to a wide range of drugs are excluded to avoid the risk of complications during the study.
Patients who have recently undergone immunomodulatory treatments (TNF inhibitors, interferons) are excluded to prevent influences on systemic inflammation.
Patients with severe cognitive impairment or psychiatric conditions that could affect the ability to understand or comply with the study protocol are excluded.
2.3. Statistical Analysis
The statistical analysis was performed using MedCalc Statistical Software (Version number 20.0), leveraging its specialized functionalities for hypothesis testing, regression modeling, and data visualization. Continuous variables were summarized as means with standard deviations, while categorical variables were expressed as frequencies and percentages. The selection of statistical methods was guided by the distribution and nature of the data, as determined through the rigorous testing of assumptions.
To assess the distribution of continuous variables, normality was tested using the Shapiro–Wilk test, while the homogeneity of variances was evaluated using Levene’s test. For variables such as viral load, which exhibited a highly skewed distribution, a Log10 transformation was applied to achieve normality. Post-transformation, normality, and homoscedasticity were reassessed to ensure the validity of subsequent statistical analyses. For variables that failed these assumptions even after transformation (e.g., biomarkers with high variability), non-parametric methods such as the Kruskal–Wallis test were employed as alternatives to the ANOVA.
For continuous variables meeting parametric assumptions, a one-way ANOVA was used to compare means across the three groups, followed by post hoc analyses to identify significant pairwise differences. Categorical variables were analyzed using the chi-square test to evaluate proportion differences between groups. For variables with small, expected cell counts, Fisher’s exact test was used to ensure accurate results.
Relationships between inflammatory biomarkers and gastrointestinal symptoms were examined using Pearson correlation coefficients for linear associations. Spearman’s rank correlation was used as a robust, non-parametric alternative for variables or relationships that violated linearity or normality assumptions.
Multivariate logistic regression was utilized to identify independent predictors of gastrointestinal symptoms, with the dependent variable being a binary outcome indicating the presence or absence of gastrointestinal symptoms 6 months after antiretroviral therapy (ART). Predictor variables, including inflammatory biomarkers (e.g., CRP, IL-6, TNF-α), age, and BMI, were chosen based on their clinical relevance and supporting evidence from previous studies. Collinearity diagnostics were conducted to ensure the model’s validity, confirming the independence of the predictor variables. Additionally, Bonferroni corrections were applied to control for the family-wise error rate, and post hoc multiple comparisons were conducted where appropriate.
Missing data were addressed through appropriate methods: listwise deletion was used for variables with a small percentage of missing values (<5%). At the same time, multiple imputation was applied for variables with more substantial gaps to ensure robust analyses.
A significance threshold of p < 0.05 was used for all tests, except where adjustments for multiple comparisons were necessary. The analytical approach was designed to account for potential confounding variables and ensure reproducibility and rigor in interpreting results.
By systematically applying these statistical methods, the study ensured that the findings were both reliable and scientifically robust, offering valid insights into the relationships between inflammatory biomarkers, gastrointestinal outcomes, and group characteristics.
2.4. Ethical Considerations
The patient’s medical records were securely maintained in a database compliant with data protection laws. Access to these records was granted only after obtaining informed consent from the patients, safeguarding their confidentiality, and ensuring their rights were respected.
The procedures carried out in this research adhered strictly to the guidelines set by the relevant human experimentation committee at both institutional and national levels. They were also aligned with the principles of the Helsinki Declaration of 1975, as revised in 2013. Informed consent was obtained from each patient included in the study, ensuring they were thoroughly informed about the nature and scope of the research.
This research was approved by the hospital’s Ethics Committee, with approval number 8947/28 September 2018. This approval verified that the study adhered to all pertinent ethical guidelines, ensuring the protection and respect of all human participants involved in the research.
3. Results
The findings demonstrate that elevated levels of CRP, IL-6, TNF-α, and fibrinogen are all significant predictors of gastrointestinal symptoms, as indicated by their high OR values and narrow confidence intervals. For instance, TNF-α exhibited an OR of 1.82 (95% CI: 1.45–2.31, p < 0.0001), underscoring its strong association with gastrointestinal side effects. Similarly, markers such as CRP (OR 1.75) and IL-6 (OR 1.68) also show a significant predictive value for the occurrence of these symptoms.
In addition, IL-1β, IFN-γ, and D-dimer emerged as independent predictors of gastrointestinal complications, reinforcing the critical role of systemic inflammation in developing these adverse effects, particularly in patients with HIV and type II diabetes.
4. Discussion
The results of this study provide valuable insights into the complex relationship between systemic inflammation, gastrointestinal side effects, and the impact of antiretroviral therapy (ART) in HIV-positive patients, particularly those with type II diabetes. By analyzing inflammatory biomarkers and their association with gastrointestinal symptoms, this research not only aligns with the existing literature but also highlights the urgent need for tailored therapeutic strategies to optimize patient outcomes.
Addressing systemic inflammation and its gastrointestinal consequences is essential to improving ART adherence and quality of life in HIV-positive patients. Severe gastrointestinal symptoms, in particular, significantly impact daily activities and can lead to suboptimal ART adherence, jeopardizing long-term viral suppression and overall patient outcomes.
Future research directions should include longitudinal studies spanning 12–24 months to evaluate the long-term impact of systemic inflammation on gastrointestinal symptoms and comorbidities, providing opportunities to identify optimal timing for therapeutic interventions. Investigating biomarkers as predictive tools for gastrointestinal and systemic complications, alongside establishing critical thresholds for guiding early interventions, is a priority. Additionally, clinical studies exploring the efficacy of adjuvant therapies, such as probiotics and anti-inflammatory agents, could help reduce inflammation and improve gastrointestinal outcomes. Integrating precision medicine approaches, including genetic profiling and individualized ART regimens, could optimize care by tailoring treatments to the specific needs of patients based on genetic and metabolic factors.
A significant and innovative contribution of this study lies in its integrated evaluation of inflammatory biomarkers and gastrointestinal symptoms in HIV-positive patients, with a special focus on the influence of type II diabetes. While many studies have separately investigated ART-associated inflammation or gastrointestinal side effects, our research is among the few to explore the complex interplay between these variables in a comparative framework that includes both HIV-positive patients without diabetes and those with type II diabetes. Furthermore, the use of standardized ART regimens based on new-generation medications minimizes variability and allows for a clearer interpretation of relationships between inflammation, comorbidities, and symptoms. The uniform six-month reevaluation period adds a unique methodological dimension, capturing ART effects after treatment stabilization. This approach not only provides a detailed perspective on inflammatory and gastrointestinal processes but also identifies key intervention points, underscoring the need for personalized therapeutic strategies, an aspect insufficiently explored in the existing literature.
Strengths and Limitations
This study offers several notable strengths. Firstly, it provides new insights into the correlation between elevated inflammatory biomarkers—such as CRP, IL-6, TNF-α, and fibrinogen—and gastrointestinal symptoms in HIV-positive patients on ART, particularly those with type II diabetes. This novel association enriches the existing literature on HIV treatment and its side effects, emphasizing the role of systemic inflammation in gastrointestinal dysfunction. The longitudinal design further enhanced the study’s strength, which involved collecting inflammatory biomarkers both before and 6 months after ART initiation. This allowed for a dynamic evaluation of how systemic inflammation evolves with treatment and how it may contribute to gastrointestinal symptoms over time. Secondly, the comprehensive analysis of a broad range of biomarkers, including D-dimer and IL-1β, allows for a detailed evaluation of the mechanisms behind ART-related side effects, particularly in patients with comorbid conditions like type II diabetes. This thorough biomarker profiling enhances the robustness of the findings and provides a more nuanced understanding of the interplay between inflammation, ART, and gastrointestinal outcomes. Lastly, the study holds significant clinical relevance by highlighting the need for personalized treatment approaches, particularly for patients with elevated inflammatory markers, which could directly impact clinical practice. The improved management of gastrointestinal symptoms could lead to better treatment adherence and enhanced quality of life for HIV-positive patients, especially those with diabetes.
However, this study also has certain limitations. While the longitudinal design offers advantages, the observational nature of this study still limits the ability to establish definitive causality between elevated biomarkers and gastrointestinal symptoms. The six-month timeframe, while critical for observing early ART-related changes, may not fully capture the long-term dynamics of immune recovery, viral suppression, and the establishment of HIV reservoirs in various compartments of the body. Extending the follow-up period in future research would allow a deeper understanding of how systemic inflammation and gastrointestinal outcomes evolve. Additionally, while viral load approached undetectable levels for most participants and CD4+ T cell counts improved, it remains unclear whether all patients achieved their set point levels within six months. These factors underscore the need for more granular analyses of each patient’s stage in their HIV journey.
Expanding this study into a more significant, multi-center cohort would provide more comprehensive insights into how inflammation evolves with ART over extended periods and how it contributes to long-term health outcomes. Additionally, this study was conducted at a single center, which may limit the generalizability of the results to a broader population. A multi-center approach would enhance the external validity of the findings and provide a more diverse patient population for analysis. Lastly, while the study includes 240 participants, future research with larger sample sizes must confirm these findings and improve statistical power, particularly when analyzing subgroups such as those with type II diabetes.
5. Conclusions
The findings of this study indicate that, six months after initiating antiretroviral therapy (ART), HIV-positive patients with type II diabetes exhibited significantly elevated levels of systemic inflammatory markers, including CRP, IL-6, TNF-α, and fibrinogen, compared to both HIV-positive patients without diabetes and the control group. This persistent inflammation, more pronounced in those with type II diabetes, suggests that this comorbidity may contribute to chronic immune activation despite effective viral suppression achieved through ART. Additionally, chronic inflammation was associated with a higher prevalence of gastrointestinal side effects, such as nausea, diarrhea, and abdominal pain, which may negatively impact treatment adherence and quality of life.
These results highlight the potential importance of routine inflammation monitoring and personalized treatment adjustments for HIV-positive patients, particularly those with type II diabetes, to address gastrointestinal symptoms and reduce the risk of long-term non-AIDS-related complications. However, further longitudinal studies are needed to confirm these associations and explore the causal mechanisms underlying the observed relationships between systemic inflammation, ART, and gastrointestinal outcomes.
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Madalina-Ianca Suba www.mdpi.com
