Randomized Study to Evaluate the Impact of Dietary Optimization on Metabolic Profile, Immunoactivation and Cardiovascular Risk in HIV Population on ART
Phase: NA
Conditions: HIV Infection, Inflammation, Cardiovascular Risk, Mediterranean Diet
Sponsor: Parc de Salut Mar

 
 
 
PROTOCOL OF CLINICAL RESEARCH 
 
 
 
 
 
Version of the protocol and 
date 
Version: 1.2  date: 06/15/2024 
 
Code of the protocol 
 
Qualification abbreviated 
either acronym 
VIHMET 
Promoter 
 
CRO (when proceed) 
If applicable 
 
 
 
 
 
 
 
 
 
 
 
Research Team 
Principal Investigator 
Robert Güerri-Fernandez 
HIV Division. Internal Medicine. 
 Hospital del Mar 
Tel: 932483251 
e-mail: rguerri@imim.es 
Researcher(s) contributor(s) 
Juan Jose Chillaron  
Endrocinology 
Hospital del Mar, 
Barcelona  
 Tel: 932483251 
e-mail:jchillaron@psmar.cat 
Maria Esperanza Cañas-Ruano 
HIV Division. Internal Medicine. 
Tel: 932483251 
E-mail: mariadelaesperanza.canas.ruano@psmar.cat 
 
Silvia Ballesta 
Endocrinology 
Hospital del Mar 
Tel.: 932483251 
 
Randomized Study to Assess the Impact of Mediterranean Diet Optimization on 
Metabolic Profile, Immune Activation, and Cardiovascular Risk in People with HIV 
on ART 

Página 2 de 26 
 
 
E-mail: sballesta@psmarcat 
 
Itziar Arrieta Aldea  
HIV Division. Internal 
Medicine.  
 Hospital del Mar 
Tel 932483251 
E-mail: iarrieta@psmar.cat 
 
Alicia Gonzalez Mena  
HIV Division. Internal Medicine.  
Hospital del Mar. 
Tel. 932483251 
E-mail: agonzalez@psmar.cat 
 
 

Página 3 de 26 
 
SUMMARY 
 
 
 
Qualification 
Randomized Study to Assess the Impact of Mediterranean Diet 
Optimization on Metabolic Profile, Immune Activation, and Cardiovascular 
Risk in People with HIV on ART 
 
 
 
 
 
 
 
Goals 
- 
Assess changes in he profile metabolic and lipid in the week 24 
- 
immunoactivation profile at week 24 
- 
To assess changes in arterial stiffness levels measured by pulse 
wave velocity at week 24 
- 
Proportion of participants with >20% decrease in TMAO in week 24 
compared to baseline. 
- 
Proportion of participants with an increase of >2 points in the 
MEDAS scale of adherence to the Mediterranean diet at week 24 
- 
Proportion of participants improving on the EQ-5D quality of life 
questionnaire at week 24 
- 
Assess persistence of all changes from Objective 1 to 6 at Week 48 
- 
To 
evaluate 
the 
presence 
and 
composition 
of 
coronary 
atheromatous plaque by means of coronary CT angiography before 
and after the intervention. 
 
Design 
Rehearsal clinical randomized open 
 
 
 
 
 
 
 
Inclusion-
exclusion 
criteria 
Criteria of inclusion: 
- 
Participant ≥ 18 years. 
- 
Infection by HIV confirmed by serology. 
- 
Treatment antiretroviral asset (TAR). 
- 
Burden viral undetectable during to the less 12 months (HE allows 
a blip below 200 copies). 
- 
LDL >140 mg/ dL . 
- 
Mediterranean Diet Adherence Screener (MEDAS) less than 9 
points (adherence low) for ease the detection of changes after the 
intervention. 
Criteria of exclusion: 
- 
Participants in treatment lipid-lowering. 
- 
Treatment with anti-inflammatory chronic. 
- 
Hepatitis B, C or Others conditions inflammatory chronicles active. 
- 
Hypercholesterolemia familiar No treated. 
- 
Hypothyroidism No controlled. 
- 
Impossibility of give he consent. 
Treatments 
or interventions Advice dietary, analysis blood, measurement of the vibe arterial pulse 
 
 
 
 
Variables 
Cholesterol total, cLDL,cHDL , triglycerides 
Number, size and composition of particles of lipoproteins 
Anthropometric profile 
CD 3,CD45,CD4,CD8,CCR7,CXCR3,CCR6,CD103,FOXP3,CD25,CD45RA 
and CD45RO 
IL-6, TNF-alpha, IFN-gamma, sCD163, sCD14, MADCAM-1, sCD14 and 
LPS. 
Rigidity arterial extent by the speed of vibe of pulse 
Trimethylamine -N-Oxide 
Scale MEDAS of adherence to the diet Mediterranean 

Página 4 de 26 
 
Atheroma plaque (number of plaques) 
Plaque composition (% fibrosis, % calcium, % fat) 
Coronary calcium index 
 
Questionnaire of quality of life EQ- 5D 
Population and 
No. subjects 
100 patients of the unit of HIV 
Participating 
centers 
Hospital of the Sea. Barcelona 
Study 
Description 
Study comparative of the intervention dietetics conventional with a 
personalized dietary intervention 
Overall 
duration 
from the 
study 
 
January 2025- December 2026 
Duration of the 
study for the 
patient 
48 weeks 

Página 5 de 26 
 
Board 1. Calendar of the study / procedures for a subject 
 
 
Screening 
( max 28 
days before 
baseline 
visit) 
visit ( 
randomization ) 
W4 
(+/- 7d) 
W12 
(+/-7d) 
W24 
(+/-7d) 
W48 
(+14-7d) 
Type of visit 
Intervention 
group 
Cluster control 
In person 
In person 
 
Virtual 
No 
visit 
 
Virtual 
No 
visit 
In person 
In person 
Informed consent 
x 
 
 
 
 
 
Criteria  
of 
inclusion 
 
a
nd exclusion 
x 
 
 
 
 
 
Demographic 
data 
x 
 
 
 
 
 
History medical 
x 
 
 
 
 
 
Related story 
with HIV 
x 
 
 
 
 
 
Exam physical 
 
x 
 
 
x 
x 
Anthropometric 
data 
 
x 
 
 
x 
x 
Questionnaires 
(MEDAS,   FFQ, 
SMAQ,  
EQ- 
5D 
and IPAQ) 
X  
(only 
MEDAS) 
x 
 
 
x 
x 
Concomitant 
medication 
x 
x 
 
x 
x 
x 
Adverse 
effects 
 
x 
 
x 
x 
x 
Test  
of 
pregnancy 
 
x 
 
 
x 
x 

Página 6 de 26 
 
if applicable 
 
 
 
 
 
 
Analytical studies 
Valid 
available last 
 
28 
days 
x 
 
 
x 
x 
Sample storage
 
 
 
x 
 
 
x 
x 
Pulse wave 
velocity 
 
x 
 
 
X 
 
Interview 
 
wit
h nutritionist 
 
x 
x* 
x* 
x 
 
Noninvasive 
coronary artery 
angiography 
using CT (CCTA) 
 
 
x 
 
 
 
x 
 
*Only cluster intervention 

Página 7 de 26 
 
INDEX 
 
1. List of abbreviations 
 
2. History of versions 
3. Frame theorist 
 
3.1. Background and justification of the study 
3.2. Hypothesis 
 
4. Goals 
5. Methodology 
5.1. Design of study 
 
5.2. Study population, inclusion/exclusion criteria and subject withdrawal criteria 
5.3. Definition of the variables of study 
6. Procedures of the study 
7. Period of the study 
 
8. Methods statisticians 
9. Aspects ethical 
 
10. Management of samples 
11. Confidentiality of the data 
12. Literature 
 
13. Annexes 

Página 8 de 26 
 
 
1. LIST OF ABBREVIATIONS 
 
HIV 
Virus of the immunodeficiency human 
PLHIV 
Person that lives with he HIV 
TAR 
Treatment antiretroviral 
AIDS 
Syndrome of immunodeficiency acquired human 
LDL 
Lipoproteins of low density 
MRI 
Resonance magnetic nuclear 
TMAO 
N-oxide of trimethylamine 
 
 
2. HISTORY OF VERSIONS 
 
Version 
Date 
Description of the change 
Brief justification 
1.2 
June 15, 2024 
Introduction to the amendment 
Noninvasive coronary artery 
angiography using CT (CCTA) 
 
Given the possibility of having CCTA 
experience for coronary plaque detection 
at our center, this amendment is included 
to evaluate subclinical coronary disease 
and possible changes. 
 
3. FRAME THEORIST 
3.1  BACKGROUND AND JUSTIFICATION OF THE STUDY 
 
Since the implementation of highly active antiretroviral therapy (ART), HIV-related 
mortality has been dramatically reduced. Morbidity and mortality are no longer caused 
by acquired immunodeficiency syndrome. Liver disease, cardiovascular disease, and 
non-AIDS-defining malignancies are the main causes of death. Compared with the 
general population, people living with HIV (PLHIV) have some degree of chronic immune 
activation leading to accelerated immune senescence. This process may drive the 
development of more non-AIDS-related comorbidities compared with uninfected 
individuals. By it so much, the PLHIV develop further events cardiovascular (MACE). 
Finding new targets to reduce cardiovascular risk is critical to decreasing MACE and 
improving the quality of life of PLHIV. Multiple mechanisms may drive these non-AIDS-
related events, such as chronic immune activation, inflammation, and treatment side 
effects. ART use has been associated with metabolic complications such as 
dyslipidemia, endurance to the insulin and distribution altered of the fat. Besides, These 
metabolic complications have been linked to an increased risk of myocardial infarction 
and cardiovascular disease. Different types of ART appear to have a different impact on 
the metabolic/lipid profile. 
 
Diet also has a role in metabolic control independently of ART. The impact of dietary 
intake of saturated fat and cholesterol on lipids The relationship between serum 
cholesterol and subsequent development of cardiovascular disease has been 
consistently demonstrated. On the contrary, the degree of dietary fat and cholesterol 
intake, the composition of that fat and the interaction of ART in PLHIV have an impact a 
stranger in the levels of lipids serum. Several studies previous to 

Página 9 de 26 
 
Small-scale studies in HIV-infected individuals demonstrated no relationship between 
saturated fat intake and serum lipid levels. 
Profile lipid 
HIV infection is known to be associated with a highly atherogenic pattern characterized 
by increased levels and reduced size of low-density lipoproteins (LDL) and decreased 
levels of high-density lipoproteins. The key factor in the relationship between he HIV and 
the dyslipidemia go on without stay up late. The infection by HIV and ART are probably 
involved. In any case, the consequence of this persistent dyslipidemia could increase 
cardiovascular risk and the progression of atherosclerosis, which in addition to persistent 
inflammation could increase MACE. The standardization of the determination of LDL 
cholesterol ( cLDL ) is technically complex. This fact, together with the variability of some 
lipid components between individuals, has generated difficulties in the interpretation of 
the results of the lipid profile and its association with cardiovascular risk. The 
conventional methods of lipoprotein quantification used in clinical laboratories only allow 
the quantification of concentrations. Therefore, it is necessary to find new techniques 
capable of estimating cardiovascular risk with greater precision. This requires the ability 
to distinguish between different classes and subclasses of lipoproteins and to quantify 
particle size using nuclear magnetic resonance (NMR) spectroscopy. NMR has been 
shown to be a more accurate technique in different types of population. To date, few 
studies have been conducted using NMR in people living with HIV. With NMR we can 
estimate with precision he risk cardiovascular to the identify and quantify different types 
of lipoproteins according to their physicochemical characteristics, which allows 
determining with greater precision and accuracy the degree of penetration of these 
particles into the endothelium vascular. This technique can allow us have a better 
comprehension of quantitative and qualitative changes in lipoprotein profiles. Some 
studies explored the relationship between lipoprotein subclass profiles assessed by MRI 
and subclinical atherosclerosis by noninvasive means, such as intima-media thickness 
or aortic pulse wave velocity. 
 
Speed of vibe of pulse 
Arterial stiffness may lead to the development of vascular complications and premature 
vascular aging . It is a reliable biomarker of cardiovascular damage. Aortic pulse wave 
velocity is an indicator of aortic stiffness and is used as a predictor of adverse 
cardiovascular events. In addition, this technique is able to detect changes before intima-
media thickness. Data on arterial pulse wave velocity in PLHIV are scarce. Since it is a 
simple, noninvasive technique, it could be useful to assess cardiovascular risk in this 
population. 
Biomarkers metabolic of risk cardiovascular 
Trimethylamine N-oxide (TMAO) is a gut microbiota-dependent metabolite generated 
from the oxidation of trimethylamine . TMAO is an independent cardiovascular factor 
related to inflammation, oxidative stress, cardiac fibrosis, endothelial injury, 
hypertension, and platelet activation. PLWH are potentially more susceptible to TMAO 
due to increased intestinal permeability. Increased intestinal permeability would increase 
the translocation of this bacterial product and, consequently, increase immune activation 
and inflammatory cytokine expression. The Diet has an impact on TMAO. Vegetarians 
have reduced TMAO levels 

Página 10 de 26 
 
while people who did not follow the Mediterranean diet had increased levels according 
to two studies. Dietary changes in TMAO measurements have not been investigated 
before in HIV infection. Acting on the intestinal microbiota and the diet could be a via 
potential for reduce he risk cardiovascular in PLWH. Any impact on diet and lifestyle 
could change the TMAO profile and could be considered as an indirect marker of 
cardiovascular disease. in a via different to the metabolism lipid. His measurement could 
provide additional information on lipids and be an outcome of interest to evaluate the 
result of a dietary intervention. 
 
Risk cardiovascular and activation immunological 
Previous studies in HIV have separately associated subclinical atherosclerosis with 
markers serum of inflammation, activation widespread of cells T, activation of 
cytomegalovirus-specific T cells and soluble CD163 (a marker of monocyte activation). 
The role of T cells and the adaptive immune response in atherosclerosis has long been 
known. Although murine models suggested that CD4+ cells may be more atherogenic 
than CD8+ cells within plaques, circulating CD8+ cells can also promote atherosclerosis 
in response to activation by intracellular infections such as cytomegalovirus or Chlamydia 
pneumoniae . Kaplan et al have shown that in HIV, CD8+ activation may be more 
predictive of carotid plaque whereas CD4+ activation is more closely associated with 
carotid stiffness, another measure of disease vascular subclinical. All the data clinical 
published until he moment suggest that exists a narrow relationship between the 
activation immunological derivative of HIV infection and the extent of cardiovascular 
damage. 
Diet Mediterranean in PLHIV 
The Mediterranean diet, supplemented with virgin olive oil and nuts, has shown, in people 
at high cardiovascular risk, a reduction in MACE in primary and secondary prevention. 
The Mediterranean diet has also been associated with a reduction in age-related 
inflammation. Stradling et al. conducted a feasibility study in PLWH who adopted a 
Mediterranean portfolio diet supplemented with with walnuts reducers of fat. The results 
They suggested improvements in blood pressure, LDL cholesterol, and diet quality after 
6 months. Another study in PLWH showed improvement in metabolic parameters, 
immune activation, regulatory T cell subsets, and microbiota . 
 
Noninvasive coronary artery angiography using CT (CCTA) 
 
Noninvasive coronary angiography using computed tomography allows a detailed and 
noninvasive assessment of the coronary arteries, their anatomy, the presence of 
atherosclerotic plaques, their location and level of severity , and the presence of 
qualitative characteristics in the plaques that increase their risk of rupture. Currently, 
CCTA can be performed with very low doses of radiation (around 3 mSv), and the total 
time of the test is just a few minutes. The test requires the use of intravenous contrast 
for a better definition of the coronary lumen, although relevant complications associated 
with the use of contrast are very rare. Due to its advantages, CCTA has become a key 
test in the assessment of patients with stable chest pain, it is recommended as a class I 
test by the guidelines of the European Society of Cardiology (), and its clinical use is 
increasing rapidly in our environment. 
 
 
 
 
 
In conclusion, the Mediterranean diet in the general population with cardiovascular risk 
factors reduced the incidence of major adverse cardiovascular events. In PLWH, the diet 
Mediterranean ha shown some improvements immune and metabolic. Reduce he risk 

Página 11 de 26 
 
cardiovascular associated to the HIV is at the moment one of the Main objectives of the 
investigation in HIV. HE they have identified several techniques for estimate the risk, but 
we do not yet have an adequate tool to reduce it. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Página 12 de 26 
 
 
3.HYPOTHESIS 
This study HE base in the following hypothesis: 
1. HIV infection and ART negatively impact the lipid profile of people living with HIV. 
2. HIV infection is associated with an increase in cardiovascular disease through 
different pathways (classical risk factors, persistent inflammation, antiretroviral 
treatment, etc. ). 

Página 13 de 26 
 
3. The infection HIV HE associates to a profile lipid abnormal so much of the cholesterol 
total, HDL, LDL and lipoprotein subclasses measured by proton magnetic resonance 
imaging. 
4. HIV infection drives early atherosclerosis mediated, in part, by an adverse lipid profile 
and persistent immune activation. 
5. Modifying the diet towards a Mediterranean diet will produce changes in the lipid 
profile, in immunoactivation markers , in inflammatory parameters, in clinical markers of 
subclinical atherosclerotic disease such as the pulse wave and in markers of metabolites 
associated with cardiovascular disease such as serum Trimethylamine -N-Oxide 
(TMAO). 
4. Objectives 
Based in the previous hypothesis We propose the following Objectives: 
Objective 1. Evaluate changes in the metabolic and lipid profile: 
- 
Objective 1a. To evaluate changes in the lipid profile (total cholesterol, LDL-C , 
HDL-C , triglycerides) at week 24 compared to baseline. 
- 
Objective 1b To evaluate the number, size and composition of lipoprotein 
particles by proton magnetic resonance spectroscopy at week 24 compared to 
baseline. 
- 
Objective 1c. Percentage of people with increased high-density lipoprotein (HDL) 
levels >5%. 
- 
Objective 1d. To assess changes in the anthropometric profile (weight, body 
mass index, abdominal circumference) at week 24 compared to baseline. 
Aim 2 . Assess changes in he profile inflammatory and of immunoactivation : 
- 
Aim 2a. Assess the changes in the activation immune of the T cell compartment 
at week 24 compared to baseline. 
- 
Objective 2b. Percentage of patients with reduced inflammatory and bacterial 
translocation markers at week 24 compared to baseline. 
Objective 3. To evaluate changes in arterial stiffness levels measured by pulse wave 
velocity at week 24 compared to baseline. 
Aim 4 . Proportion of Participants with decline of >20% of TMAO in the week 24 
compared to baseline. 
Aim 5 .Proportion of Participants with increase of >2 points in the scale MEDAS 
adherence to the Mediterranean diet at week 24 compared to baseline 
Objective 6. Proportion of participants who improve in the quality of life questionnaire. 
EQ-5D life at week 24. 
Objective 7. Evaluate the persistence of all changes included in objectives 1 to 6 at 
week 48 (24 weeks after the end of the intervention) 
Objective 8. To assess the burden of subclinical atherosclerotic coronary disease and 
changes 1 year after intervention. 
 
 
 
 
 
 
 
 
 
 
 
 
 

Página 14 de 26 
 
5. METHODOLOGY 
 
5.1. 
Design of study 
Study randomized prospective of intervention with optimization dietetics in 
people with HIV in treatment antiretroviral with dyslipidemia (Cholesterol total 
>200mg/dl either LDL cholesterol >160mg/dl) and without treatment lipid-lowering. 
5.2. 
Study population, inclusion/exclusion criteria and subject withdrawal criteria. 
Criteria of inclusion: 
- 
Participant ≥ 18 years. 
- 
Infection by HIV confirmed by serology. 
- 
Treatment antiretroviral asset (TAR). 

Página 15 de 26 
 
- 
Burden viral undetectable during to the less 12 months (HE allows a blip 
below 200 copies). 
- 
LDL >140 mg/ dL . 
- 
Mediterranean Diet Adherence Screener (MEDAS) lower to 9 points 
(low adhesion) to facilitate the detection of changes after the intervention. 
 
Criteria of exclusion: 
- 
Participants in treatment lipid-lowering. 
- 
Treatment with anti-inflammatory chronic. 
- 
Hepatitis B, C or Others conditions inflammatory chronicles active. 
- 
Hypercholesterolemia familiar No treated. 
- 
Hypothyroidism No controlled. 
- 
Impossibility of give he consent. 
Criteria of withdrawal: 
Subjects may withdraw from the study at any time without obligation to provide a reason. 
He IP can give of low to a subject of the study by reasons of safety or efficacy. 
The reasons of abandonment of the study will be the following: 
- Failed of screening: All patient that, a time informed is spoiled in 
participating in the study, does not meet the inclusion and/or exclusion criteria. 
- Withdrawal of the consent: All patient that, by any reason, remove your consent 
- Consumption of anti-inflammatory either lipid-lowering drugs during further of 7 days. 
- Diagnosis of hypothyroidism either diabetes mellitus. 
- Any event adverse that prevent continue any diet indicated. 
- Loss of follow-up. 
 
5.3. 
Definition of the variables of study 
 
Main Variables 
- 
Total cholesterol, cLDL , cHDL , triglycerides in mg/ dL . It will be measured at 
baseline, week 24 and 48. 
Secondary Variables 
- 
Number, size and composition of lipoprotein particles by proton magnetic 
resonance spectroscopy at baseline, week 24 and 48. 
- 
Anthropometric profile (weight, body mass index, abdominal circumference) at 
baseline, week 24 and 48. 
- 
CD 3,CD 45,CD4,CD8,CCR7,CXCR3,CCR6,CD103,FOXP3,CD25,CD45RA  
and 
CD45RO. Quantification in blood in visits basal, 24 weeks and 48 weeks of cells 
with markers. 
- 
IL-6, TNF-alpha, IFN-gamma, sCD163, sCD14, MADCAM-1, sCD14 and LPS. 
Quantification in serum in visit basal, 24 weeks and 48 weeks of 
- 
Rigidity arterial extent by the speed of vibe of pulse in the visit basal, week 
24 and 48. 
- 
Levels of Trimethylamine -N-Oxide serum (TMAO) in the in the visit basal, 
week 24 and 48. 

Página 16 de 26 
 
- 
Scale MEDAS of adherence to the diet Mediterranean measures in the 
Baseline visit, week 24 and 48 
- 
Questionnaire of quality of life EQ-5D in the visit basal, week 24 and 48. 
- 
Presence of atheromatous plaque at the coronary level at the baseline visit and at week 48 
- 
Presence of calcium, fibrous and fatty fraction in the coronary plaque at the baseline visit and at 
week 48 
- 
 
 
 
6. PROCEDURES OF THE STUDY 
• 
Pre-screening visit (Recruitment ): carried out by the regular doctor in the 
outpatient clinics of the HIV unit at Hospital del Mar. Potential candidates who 
meet the inclusion criteria will be identified. They will be given an appointment 
for: Initial visit: informed consent will be signed, blood tests will be performed with 
blood samples (to achieve objectives 1 to 6). A comprehensive history of clinical 
history, HIV infection, and drug history will be taken. Randomization will be 
carried out following randomization tables according to age, gender, and ART. 
They will be referred to an intervention consultation. 
• 
Visit 1 (baseline) : anthropometric measurements will be taken, scales 
predetermined in the study (annexes) will be passed and the measurement of the 
pulse wave velocity (according to methods described later). A dietary approach 
will then be carried out with a different visit schedule: 
*Cluster Intervention (67 participants): 
The intervention group will receive at the beginning a motivational interview with 
a personalized portfolio provided by a nutritionist on the Mediterranean diet and 
adherence to the diet: with information on the food components; desirable 
frequency of intake; recipes, use of olive oil, walnuts, hazelnuts and almonds. 
Individual guidelines will be planned individually to improve the lipid profile and 
improve food choices considering dietary habits, socioeconomic status and 
nutritional needs (and the score obtained on the MEDAS scale). 
At weeks 4 and 12 (virtual), the patient will have a new interview with the 
nutritionist to review the recommendations, discuss difficulties with diet 
adherence, design weekly dietary plans and shopping lists. At week 24 and 48, 
an in-person visit will be held to perform all the procedures of the baseline visit 
again (including blood draws ). 
*Cluster treatment standard (33 participants). 
Baseline visit (in person), In addition to the initial assessment identical to that of 
the intervention group, general dietary information will be provided in writing, as 
is currently done in the HIV unit. 
New visits will be made at week 24 (in person) and week 48 (in person). The 
same measurements, dietary assessments and blood draws will be made as at 
the baseline visit. 
Detailing of tasks to carry out for achievement of the goals: 
• 
Task 1. Adherence to the Mediterranean diet, anthropometric and quality of life 
measurements (objectives 5 and objective 6). All carried out by a trained 
researcher. 
- 
Task 1a. 118-item semiquantitative food frequency questionnaire (FFQ), 
MEDAS questionnaire, and a 24-hour dietary recall . 
- 
Task 1b. Anthropometric measurements: height and weight will be 
measured using a mechanical anthropometric scale. BMI (BMI = weight 
(kg) / [height (cm)]2). Waist circumference halfway between the lower rib 
margin 

Página 17 de 26 
 
and the iliac crest. Hip circumference will be measured as the widest 
measurement over the buttocks. 
- 
Task 1c. Measuring quality of life using the EQ-5D questionnaire. Specific 
questions on alcohol consumption patterns and the IPAQ Physical Activity 
Questionnaire, Simplified Medication Adherence Questionnaire (SMAQ). 
• 
Task 2. Measurement of the profile lipid (for achievement of goals 1 (1a,1b,1c) 
- 
Task 2a. Blood collection after fasting for more than 8 hours, between 8am 
and 10am. Serum will be obtained for the usual clinical measurement by 
means of of the practice clinic usual and in he laboratory of Reference 
from Catalonia for the measurement of total cholesterol, HDL and LDL 
(according to standard practice). Serum, plasma and cells will be 
preserved (task 4) 
- 
Task 2b. With the samples of serum obtained and preserved to -80ºC and 
when recruitment and monitoring have been completed, the analysis of 
the lipoprotein subclass profile will be performed by means of Proton 
Nuclear Magnetic Resonance (NMR) in Metabolomics Platform IISPV, 
CIBERDEM. Rovira i Virgili University , Bisofer Teslab . Lipoprotein 
analysis of serum samples will be performed using Liposcale ® ( Biosfer 
Teslab , Reus, Spain), a method based on 2D diffusion-ordered 1H-NMR 
spectroscopy. This protocol evaluates lipid concentrations, particle size 
and number of three different classes of lipoproteins ( vLDL ), LDL and 
HDL), as well as the number of particles of nine subclasses ( large, 
medium and small vLDL , LDL and HDL). This technique records 2D 
1HRMN spectra on a BrukerAvance III 600 spectrometer, operating at a 
proton frequency of 600 MHz to 310 KHz ( Bruker (BioSpin , Rheinstetten 
, Germany). The signal obtained makes it possible to determine the nine 
lipoprotein subclasses. (J. Clin. Med . 1875).) The study of lipoprotein 
subclasses makes it possible to obtain a profile that, when compared with 
a population database, makes it possible to estimate cardiovascular risk 
based on the lipid composition of the lipoprotein subclasses in a more 
specific way than by means of the classic scales for estimating 
cardiovascular risk. 
• 
Task 3. Measurement of arterial stiffness by pulse wave velocity (to achieve 
objective 3). Arterial pulse wave velocity will be determined by sequential 
applanation tonometry (Millar Tonometer, SPC-301; Millar Instruments, Houston, 
TX) in the carotid and femoral arteries synchronized with a three-lead 
electrocardiogram using the SphygmoCor device ( SphygmoCor ; AtCor , Sydney 
, Australia). Wave arrival latency time will be calculated using a foot-wave 
method. The surface distance from the suprasternal notch to each recording point 
will be measured. Total transit distance will be calculated by subtracting the 
distance between the sternal notch and the carotid from the distance between 
the sternal notch and the femoral artery. Arterial pulse wave velocity will be 
calculated using total transit distance divided by time. of latency. HE will reject 
the measurements of the speed of the vibe pulse that do not meet the automatic 
quality controls specified by SphygmoCor software . Two pulse wave velocity 
measurements will be averaged for each subject for all calculations. 
• 
Task 4. Study of immunoactivation markers in PBMCs (peripheral blood 
mononuclear cells) (objective 2a). PBMCs will be obtained from heparinized 
blood by density gradient centrifugation ( Ficoll - Paque ; Amersham Biosciences 
). 

Página 18 de 26 
 
• 
Task 4a. Flow cytometry. A panel of antibodies will be used to characterize 
adaptive immune cells, including CD3, CD 45, CD 4, CD8, CCR7, CXCR3, CCR6, 
CD103, 
FOXP3, 
CD25, 
CD45RA 
 
a
nd 
CD45RO. HE will identify the cells T of memory (CD45RA-/CD45RO+) and naïve 
and effector T cells (CD45RO-/CD45RA+). Within memory cells (CD45RA-), 
central memory cells (CCR7+) and effector memory cells (CCR7-); and inside of 
the cells T that No are of memory (CD45RO- 
/CD45RA+ ), naïve cells (CCR7+) and effector cells (CCR7-). Using lineage 
marker (CD3+) and cytotoxic lymphocytes (CD8+), helper lymphocytes (CD4+) 
are separated into subtypes by expression or not of the chemokine receptors 
type 3 (CXCR3) and 6 (CCR6) such as: Th1 (CXCR3+CCR6- ),Th 2 (CXCR3- 
CCR6-),Th17 (CXCR3-CCR6+) and Th1- Th17 
(CXCR3+CCR6+ ). The percentage of activated cells will be differentiated using 
the activation markers HLA-DR and CD38. Regulatory T cells will be identified as 
FOXP3+CD25+. Appropriate positive and negative controls will be performed. 
Cell populations will be acquired from LSR- Fortessa (UPF central facilities) and 
analyzed using Flowjo software ( Treestar Inc. CA). 
• 
Task 5. Study of inflammatory markers and bacterial translocation (to achieve 
objective 2b). A multiplex ELISA for different biomarkers: at least the multiplex 
must include: IL-6, TNF-alpha, IFN-gamma, sCD163, sCD14, MADCAM-1, 
sCD14 and LPS. Plasma samples will be stored at -80 °C to perform the analysis 
at the same time in order to reduce variability. 
• 
Task 6. Determination of the levels plasmatic of TMAO (for achievement of aim 
4). HE will preserve to -80C all the samples and HE will perform a Once the study 
is completed, the determination of TMAO levels in fasting plasma will be 
quantified by stable isotope dilution liquid chromatography with on-line tandem 
mass spectrometry (LC/MS/MS) on an APi 500 triple quadruple mass 
spectrometer (AB SCIEX, Framingham, Massachusetts) available at the 
Bioanalytics department of Middlesex University , London, with whom the PI has 
a stable collaboration and will allow the measurement to be carried out reliably 
and consistent. 
• 
Task 7. Upon approval of the amendment (version 1.2 of the protocol), all enrolled 
patients will undergo a CCTA. CCTAs in the study will be performed in an identical 
manner to those performed daily in our center as part of routine clinical practice, 
using identical protocols for both image capture and interpretation/reporting . The 
radiation dose used will follow the ALARA principle (“as low as reasonably 
possible”), as low as possible while maintaining image quality. The same principle 
will apply to the intravenous contrast dose. Patients will sign an updated informed 
consent to participate in the VIHMET study (insert the name of your study), which 
will include a specific description of the CCTA, its objectives and associated risks. 
Once in the study, participants will receive the usual pre-CCTA preparation by 
specialized nursing staff and directed by cardiologists with experience in cardiac 
imaging. This includes general recommendations on hydration and fasting, as well 
as the potential use of a low-dose beta-blocker (or ivabradine ) to control heart rate 
if it is above 70 bpm . 
• 
 
 
 
 
 

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7. 
PERIOD OF THE STUDY 
 
For can carry to cape all he project it we have divided in different stages: 
1. Preparation and validation of the protocol. Patient preselection circuits and 
diagnostic tests. 
January 2025 to February 2025. 
This phase will be carried out by the researchers: Design of the patient selection 
circuit within the HIV unit and promotion of the study among the professionals of 
the unit focused on the need to address the optimization of the lipid profile of this 
group of people. We will review the processing of the samples (labeling of the 
samples, contact with the BioBank service , presentation of the study to the 
doctors for their knowledge). An anonymous registry of candidate patients will be 
created. 
2. Recruitment of patients 
From March 2025 to March 2026. It is estimated that it will take at least one year 
to recruit the 100 patients. 

Página 20 de 26 
 
Recruitment will be carried out at the Infectious Diseases Service of Hospital del 
Mar (HM). The referent will be Dr. Roberto Güerri Fernández (IP The researchers 
who will coordinate the recruitment process will be Dr. Alicia González. Patients 
who meet the inclusion criteria will be referred to a specific consultation created 
ad hoc to carry out the selection visit. This phase will be carried out 
interchangeably by Dr. Esperanza Cañas, Dr. Alicia González, Dr. Itziar Arrieta, 
Dr. Mariano Pascual or Dr. Roberto Güerri. During this visit, informed consent will 
be signed, the first sample will be extracted for the complete analysis and general 
data will be collected: background, history of HIV infection, treatment history and 
the questionnaires and the participant will be randomized. 
3. Follow-up of the patient during 48 weeks . From 
March 2025 to October 2026. 
At this stage, the specific intervention itself will be carried out. Nutritionist 
researcher Jordi Argimon together with Dr. Silvia Ballesta and Dr. Juan José 
Chillarón from the Endocrinology service will carry out a complete nutritional 
assessment. Anthropometric measurements and a measurement of the speed of 
movement will be carried out. of the vibe of pulse. HE will perform follow-up 
according to it notarized. 
4. study visits (March 2025 to October 2026). The final study visit with follow-up at 
48 weeks will be carried out in all participants. The tests contemplated in the 
protocol will be carried out. This stage will be carried out by he equipment of the 
Dr Squealer (Jordi Argimon together with the Dr. Silvia Crossbow and Dr. Juan 
Jose Chillaron ) . 
5. Sample collection and processing : blood collection and processing to obtain 
PBMCs , plasma and serum. The processing and conservation of the samples 
will be carried out in the IMIM laboratory by Dr. Güerri -Fernández and Dr. García 
Giralt. 
6. Sample preparation for FACS . Once initial recruitment is complete, preserved 
samples will be thawed, stained and prepared for FACS (Dr. Güerri-Fernández, 
Dra García-Giralt). Data reading and interpretation will be performed by Dr. 
Güerri-Fernández and an external collaborator ( Prof David M. Asmuth 
(University of California). 
7. Sample preparation and shipment to Metabolomics Platform IISPV, CIBERDEM 
. Rovira i Virgili University , Bisofer Teslab . When the follow-up is completed, 
samples will be sent ( baseline samples , 24 weeks and 48 weeks). weeks) to 
the Rovira i Virgili University for further processing and interpretation of the 
results of the lipoprotein subclasses. 
8. Preparation and shipment of plasma samples to Middlesex University , London ( 
Dr. laboratory Sha ). Once follow-up is complete, samples (baseline, 24-week 
and 48-week samples) will be sent to the Middlesex Hospital. university for the 
determination of TMAO according to the described protocol. 
9. Analysis and publication of results (from January 2026 (preliminary) to December 
2026) will be carried out by the entire research team. 
 
8. ETHICS 
The study will be  submitted  to the rules ethical of the Investigation Biomedical in 
Humans. The study will be conducted in accordance with the ethical principles derived 
from the Declaration of Helsinki (Fortaleza, Brazil, October 2013). In addition, the study 
will be conducted in accordance with the protocol, good clinical practice (GCP) in 
accordance with the guidelines of the International Conference on Harmonization (ICH) 
and the regulatory requirements for participating institutions. The study will be conducted 
according to a protocol reviewed by a Committee. The benefits of the study are 
considered to be in proportion to the risks; and the rights and well-being of the subjects 

Página 21 de 26 
 
will be respected. 
Patients will be informed and given an approved informed consent form in compliance 
with GCP. Likewise, all participants will sign the PsMar Biobank consent . Aspects related 
to confidentiality will follow established regulations. Before including subjects in the 
study, the researcher will review the information sheet with the potential participant, and 
if the participant agrees, agrees to participate, the informed consent document will be 
signed and dated. The researcher will provide a copy of the signed informed consent 
form to each subject and will keep a copy in the study file. The subject will be informed, 
as provided for in Organic Law 15/99 on the Protection of Personal Data, that these may 
be subject to automated processing and of the rights that the study participants have to 
consult, modify or delete their personal data from the file. The subject will be informed 
that the database will be handled by the IP using an access code and the name will not 
appear in the registry. The responsibility of the file global corresponds to the Institute 
Hospital of the Sea of 

Página 22 de 26 
 
Research Institute (IMIM) of Barcelona. This study will comply with the Code of Good 
Scientific Practices ( http://www.imim.es/imim/cas/c-CBPC.htm ). 
 
 
No financial compensation is expected for participants or reimbursement of expenses 
arising from their participation  
 
 
9. MANAGEMENT OF SAMPLES 
 
As previously described, during the study, blood will be drawn and processed to obtain 
PBMCs , plasma and serum. The processing and conservation of the samples will be 
carried out in the IMIM laboratory by Dr. Güerri - Fernández and Dr. García Giralt. When 
the initial recruitment is completed, the preserved samples will be thawed, stained and 
prepared to undergo FACS. When the follow-up is completed, samples will be sent ( 
baseline samples , 24 weeks and 48 weeks) to Rovira i Virgili University for further 
processing and interpretation of lipoprotein subclass results. Likewise, samples 
(baseline, 24-week and 48-week samples) will be sent to Middlesex University for TMAO 
determination. 
The samples will be pseudo-anonymized and when the study is completed, the 
remaining samples will be collected in the PSmar biobank . 
 
10. CONFIDENTIALITY OF THE DATA 
This study will be carried out in accordance with the General Data Protection Regulation 
2016/679 and Organic Law 3/2018, of December 5, on the Protection of Personal Data 
and the Guarantee of Digital Rights. 
Patient recruitment will be carried out in the outpatient clinics of the HIV unit at Hospital 
del Mar. Once the participant signs the informed consent, he or she will be assigned a 
study number that will correspond to his or her medical record number. This will be saved 
in an encrypted file with a password to which the IP will have access. 
The database that will be created ad hoc for the study will not include any data that would 
allow the patient to be identified: no date of birth, no phone number, no medical record 
number. Only the study ID. This database will be in T h e  RedCap environment will only 
be accessible to the study's researchers. 
Study data must be verifiable with the source data, which requires access to all original 
records, laboratory reports, and subject records. Confidentiality of data and patient 
identity will be maintained during and after the study is completed . Only the principal 
investigator and authorized study personnel will have access to these confidential 
records. 
None fact used in he analysis and later divulgation of the results of the study will contain 
some identifiable reference to patient names. 
 
Once the study is completed, the results will be communicated to the relevant authorities, 
as appropriate, in accordance with local legislation. The data generated will also be 
published in conferences and scientific journals. 

Página 23 de 26 
 
11. LITERATURE 
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1999 to 2011 (D ◻: A◻: D): a multicohort collaboration. Lancet. 2014;384:241 –8. 
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highly active antiretroviral therapy compared with estimates for the general population of 
similar age and from the same geographical area. HIV Med . 2007;8:251 –8. 
3. Phillips AN, and to the. The role of HIV in serious diseases other than AIDS. 
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4. TISS Group. Initiation of Antiretroviral Therapy in Early Asymptomatic HIV Infection. 
N Engl J Med . 2015;373:795 –807. 
5. Zicari S, et al. Immune activation, inflammation, and non-AIDS co-morbidities in HIV-
infected patients under long-term ART. Viruses . 2019;11. 
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insulin resistance in patients receiving HIV protease inhibitors. Aids . 1998;12:51 –8. 
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price of success. J Am Med Assoc . 2006;296:844 –54. 
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9. Lichtenstein AH. Dietary fat, carbohydrate, and protein: Effects on plasma lipoprotein 
patterns. J Lipid Res. 2006;47:1661 –7. 
10. Jackiewicz A,et to the. Effect of fatty acid content in the diet on lipid profile in 
HIV-infected patients treated with antiretroviral drugs. HIV AIDS Rev. 2019;18:25 
–32. 
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infected cohort of patients treated with protease inhibitors. HIV AIDS Rev. 
2015;14:22 –7. 
12. Rodríguez-Gallego et al. Circulating metabolomic profile can predict dyslipidemia in 
HIV patients undergoing antiretroviral therapy. Atherosclerosis . 2018;273:28 –36. 
13. Riddler SA, et al. Antiretroviral therapy is associated with an atherogenic lipoprotein 
phenotype among HIV-1-infected men in the Multicenter AIDS Cohort Study. J Acquir 
Immune Defic Syndr . 2008;48:281 –8. 
14. Korzun WJ,et al. Difference in bias approach for commutability assessment: 
Application to frozen pools of human serum measured by 8 direct methods for HDL and 
LDL cholesterol. Clin Chem . 2015;61:1107 –13. 
15. Mallol R,et al. Liposcale : A novel advanced lipoprotein test based on 2D diffusion-
ordered 1H NMR spectroscopy. J Lipid Res 2015;56:737 –46. 
16. Mallol R, et al. Human serum/plasma lipoprotein analysis by NMR: Application to the 
study of diabetic dyslipidemia. Prog Nucl Magn Reson Spectrosc . 2013;70:1 –24. 
17. Llauradó and to the. J. Clin. Med . 2019, 8(11), 1875 
18. Laurent S, Boutouyrie P. Arterial Stiffness and Hypertension in the Elderly. Front 
Cardiovasc Med . 2020;7:1 –13. 
19. Kim HL, Kim SH. Pulse Wave Velocity in Atherosclerosis. Front Cardiovasc Med . 
2019;6:1 –13. 
20. January MH, and to the. Implication of trimethylamine n-oxide (TMAO) in 
disease: Potential biomarker or new therapeutic target. Nutrients . 2018;10. 
21. Maidji E, et al. Replication of CMV in the gut of HIV-infected individuals and epithelial 
barrier dysfunction. vol. 13. 2017. 
22. Serrano-Villar S, et al. Blood Bacterial Profiles Associated With Human 
Immunodeficiency Virus Infection and Immune Recovery. J Infect Dis . 2021;223: 471–
81 . 
23. Kaplan RC, Sinclair E, Landay AL, Lurain N, Sharrett AR, Gange SJ, et al. T cell 
activation and senescence predict sub-clinical carotid artery disease in HIV-infected 
women. J Infect Dis . 2011 Feb 15;203(4):452–463 

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24. Andersson J, Libby P, Hansson GK. Adaptive immunity and atherosclerosis. Clin 
Immunol. 2010 Jan;134(1):33–46. 

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25. Kaplan RC, Sinclair E, Landay AL, Lurain N, Sharrett AR, Gange SJ, et al. T cell 
activation predicts carotid artery stiffness among HIV-infected women. Atherosclerosis. 
2011 Jul;217(1):207–213 
26. Williams K.A., et al. Nutrition Intervention for Reduction of Cardiovascular Risk in 
African Americans Using the 2019 American College of Cardiology/American Heart 
Association Primary Prevention Guidelines. Nutrients . 2021;13:3422 . 
27. Estruch R, et al. Primary Prevention of Cardiovascular Disease with a Mediterranean 
Diet Supplemented with Extra-Virgin Olive Oil or Nuts. N Engl J Med . 2018; 378:e 34. 
28. Clark JS, Simpson BS, Murphy KJ. The role of a Mediterranean diet and physical 
activity in decreasing age-related inflammation through modulation of the gut microbiota 
composition. Br J Nutr . 2021:1–16. 
29. Stradling C, et al. Randomized controlled pilot study to assess the feasibility of a 
Mediterranean Portfolio dietary intervention for cardiovascular risk reduction in HIV 
dyslipidaemia : A study protocol. BMJ Open. 2016;6:1 –11 
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management of chronic coronary syndromes. Eur Heart J. 2020;41(3):407-477

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12. ANNEXES: 
 
• 
Archives additional (exhibit MEDAS (1), EQ-5D-5L (2), SMAQ (3))