The Eswatini Study on Neurocognitive Performance in Adolescents Living With HIV

Adolescents living with HIV (ALHIV) in sub-Saharan Africa face a complex health burden that goes beyond virological control. In Eswatini, where HIV prevalence ranks among the highest globally, the combination of chronic infection, immune dysregulation, and neurodevelopmental vulnerability poses a significant public health challenge. Although antiretroviral therapy (ART) has greatly improved survival rates, emerging evidence indicates that ongoing immune activation may lead to subtle yet clinically significant neurocognitive deficits in youth. These impairments can impact academic performance, psychosocial functioning, and long-term quality of life.

This study aims to explore the biological correlates of neurocognitive performance in ALHIV, with a particular focus on inflammatory biomarkers. It seeks to determine the extent to which systemic inflammation measured through specific immunological markers relates to cognitive functioning in adolescents and whether these associations differ between HIV-positive and HIV-negative individuals.

Utilizing a case-control design, the study will generate comparative data to enhance understanding of the neuroimmune interface in this population. Study Population and Recruitment The study will enroll 80 adolescents aged 13 to 19 years, consisting of: 50 HIV-positive participants, recruited from HIV care centers at Baylor College of Medicine Children's Foundation-Eswatini (Manzini \& Mbabane), and Raleigh Fitkin Memorial Hospital. 30 HIV-negative controls, matched by age and sex, recruited from the same geographic catchment areas to ensure contextual comparability. Eligibility criteria include confirmed HIV status (positive or negative), age within the specified range, and capacity to participate in neurocognitive testing. Exclusion criteria include acute illness, neurological disorders unrelated to HIV, or inability to provide informed consent/assent.

Neurocognitive Assessment Protocol Cognitive functioning will be assessed using the Symbol Digit Modalities Test (SDMT), a standardized neuropsychological instrument sensitive to changes in processing speed and attention. The SDMT is particularly suitable for use in low-resource settings due to its minimal language dependence and brief administration time. It evaluates: Visual scanning Psychomotor speed Sustained attention Working memory Testing will be conducted in a quiet, controlled environment by trained research personnel fluent in local languages and familiar with adolescent developmental norms.

Biomarker Collection and Analysis Venous blood samples will be collected from all participants under sterile conditions. Samples will be processed and stored according to biosafety protocols and analyzed using enzyme-linked immunosorbent assay (ELISA) techniques. The following biomarkers will be quantified: C-reactive protein (CRP): A general marker of systemic inflammation. Soluble CD14 (sCD14): Reflects monocyte activation and microbial translocation. Lipopolysaccharide (LPS): Indicates gut barrier dysfunction and endotoxemia. Soluble CD163 (sCD163): Associated with macrophage activation and neurodegeneration. Monocyte chemoattractant protein-1 (MCP-1): A chemokine involved in leukocyte recruitment and neuroinflammatory signaling. These biomarkers were selected based on their relevance to HIV-associated immune activation and their potential role in neurocognitive outcomes.

Sociodemographic and Clinical Data Collection

Participants will engage in structured interviews and complete questionnaires to gather sociodemographic information, including:

• Age
• Gender
• Educational attainment
• Household composition
• Socioeconomic status

Clinical data will be sourced from medical records, encompassing:

• Duration of HIV infection
• ART regimen and adherence history
• CD4+ T-cell count and viral load
• History of opportunistic infections
• Neurological symptoms or diagnoses This comprehensive dataset will facilitate robust adjustment for confounding variables in statistical analyses.

Outcome Measures

Primary Outcome:

Neurocognitive performance scores derived from SDMT results.

Secondary Outcomes:

• Quantitative levels of inflammatory biomarkers.
• Correlation coefficients between biomarker levels and cognitive scores.
• Group differences in biomarker profiles and cognitive outcomes. Statistical Analysis Plan

Data will be analyzed using multivariate regression models to evaluate the relationship between inflammatory biomarkers and neurocognitive performance. These models will adjust for potential confounders, including:

• Age
• Sex
• Educational level
• HIV disease severity (e.g., CD4 count, viral load)
• ART adherence Interaction terms will be explored to determine if the strength of associations varies by HIV status. Additionally, structural equation modeling (SEM) will be employed to investigate potential mediating pathways, such as the role of monocyte activation in linking HIV infection to cognitive impairment. SEM allows for the simultaneous estimation of direct and indirect effects, providing a nuanced understanding of the biological mechanisms involved.

Ethical Considerations The study protocol has been reviewed and approved by the Eswatini Health and Human Research Review Board (EHHRRB). Informed consent will be obtained from all participants aged 18 and above, and assent will be obtained from minors alongside parental or guardian consent. All data will be anonymized and securely stored, with access restricted to authorized personnel. Participants will be informed of their right to withdraw at any time without consequence.

Data Management and Quality Assurance Data will be entered into a secure electronic database with built-in validation checks. Double data entry and periodic audits will be conducted to ensure accuracy. Laboratory assays will be performed in duplicate, and inter-assay variability will be monitored. All research staff will undergo training in Good Clinical Practice (GCP) and ethical conduct of research involving human subjects.

Potential Risks and Benefits Risks to participants are minimal and primarily related to blood draw procedures (e.g., discomfort, bruising). Psychological distress from cognitive testing is considered low. Participants may benefit from increased awareness of their cognitive health and receive referrals for further evaluation if significant impairments are detected.

Significance and Innovation

This study addresses a critical gap in understanding HIV-associated neurocognitive impairment in adolescents, particularly in high-prevalence, resource-limited settings. By integrating immunological and neuropsychological data, it offers a novel approach to characterizing the neuroimmune interface in ALHIV. The findings may:

• Identify biomarkers predictive of cognitive decline.
• Inform early screening and intervention strategies.
• Support the development of targeted therapies to mitigate neuroinflammation.
• Enhance clinical guidelines for adolescent HIV care. Moreover, the study contributes to global efforts to improve the long-term outcomes of adolescents living with HIV, aligning with UNAIDS goals for health equity and sustainable development.