The ILERVAS Project: Assessing the Prevalence of Subclinical Vascular Disease and Hidden Kidney Disease
The ILERVAS Project: Assessing the Prevalence of Subclinical Vascular Disease and Hidden Kidney Disease
BACKGROUND AND OBJECTIVES:

 Cardiovascular disease is the leading cause of death despite huge primary and secondary
 prevention policies with a strong economic burden. The primary objectives of the ILERVAS
 project are: (i) to identify unknown factors involved in the presence of atherosclerosis,
 metabolic syndrome, pre-diabetes and hidden kidney disease in a low/moderate cardiovascular
 risk population; (ii) to identify unknown factors involved in the progression of
 atherosclerosis, metabolic syndrome, pre-diabetes and hidden kidney disease in a low/moderate
 cardiovascular risk population; (iii) to Assess of the impact of arterial ultrasound on
 cardiovascular events and mortality in a low/moderate cardiovascular risk population.

 METHODS:

 Randomized intervention study. From 2015 to 2018, 16,660 participants (8,330 in the
 intervention group (Mobile Unit Follow-up Group) and 8,330 in the no intervention group
 (Electronic Medical History Follow-up Group )) aged between 45 and 70 years without a
 previous history of cardiovascular disease and with at least one cardiovascular risk factor
 will be randomly selected across the province of Lleida, Spain.
STUDY DESIGN AND POPULATION:

 Randomized interventional study. From 2015 to 2018, 16,660 participants (8,330 in the
 intervention group (Mobile Unit Follow-up Group) and 8,330 in the no intervention group
 (Electronic Medical History Follow-up Group)) aged between 45 and 70 years without a previous
 history of cardiovascular disease and with at least one cardiovascular risk factor will be
 randomly selected across the province of Lleida, Spain. The Ethics Committee of the
 University Hospital Arnau de Vilanova in Lleida (Spain) approved the protocol. All patients
 signed informed consent. The study was conducted according to the principles of the
 Declaration of Helsinki.

 SOURCE OF INFORMATION AND INSTRUMENTS FOR DATA COLLECTION:

 In the Mobile Unit (MU), clinical, sociodemographic and anthropometric data will be recorded.
 Patients will be evaluated with artery ultrasound (carotid, femoral, transcranial and
 abdominal aorta), ankle-brachial index, pulse wave velocity, spirometry, determination of
 advanced glycation-end products, atrial fibrillation screening, dried blood spot test and
 urine analysis. Moreover, DNA, RNA, Saliva, blood and urine samples will be collected and
 stored in the biobank to identify new biomarkers using omic studies. Additionally, climate,
 air pollutant and airborne pollen data form the entire province of Lleida will be registered.
 In addition, variables such as physical exercise, diet and daytime will be collected.

 In the no intervention group (Electronic Medical History Follow-up Group), participants will
 be followed through their electronic medical records. Sociodemographic (age, sex, race,
 marital status, education and labour status), clinical and anthropometric data and will be
 electronically collected.

 PARTICIPANT SELECTION:

 From a total of 410,246 people with an open history in the e-CAP (in October 24, 2014), the
 study population that meets the inclusion criteria will be identified. Of these, a total of
 16,660 people (8,330 in the intervention group and 8,330 in the no intervention group) will
 be selected through simple randomization without replacement, within clusters defined by
 basic healthcare area, primary care (PC) centre or doctor's office, in accordance with the
 total number of people who may be visited in the MU during one year period. The participants
 of the selected intervention group will be informed of the study objectives and the
 diagnostic tests.

 FOLLOW-UP PERIOD:

 A minimum 10-year follow-up period has been established to observe the onset of CV events. In
 the Mobile Unit Follow-up group, a second visit will be performed after four year of
 recruitment.

 CARDIOVASCULAR EVENTS AND CKD PROGRESSION:

 The onset of a CV event will be recorded according to the 10th version of the International
 Statistical Classification of Diseases (ICD-10), which includes: angina pectoris, myocardial
 infarction, transient ischemic attack, cerebrovascular accident, heart failure, arrhythmia,
 peripheral artery disease, aortic aneurysm, revascularization and angioplasty of any artery
 region. Likewise, the patient's cause of death, whether of CV origin (myocardial infarction,
 arrhythmia, heart failure, cerebrovascular accident, aortic aneurysm, mesenteric infarction
 or sudden death) or of non-CV origin (infections, cancers, accident or kidney disease) will
 be recorded. The information sources used to identify CV events will be: review of medical
 records (through the e-CAP) and consultation of the Catalonia mortality registry. CKD
 progression is defined as a doubling of creatinine or entry into renal replacement therapy,
 and will also be collected through the e-CAP.

 CLINICAL AND BIOCHEMICAL DATA:

 The following variables will be recorded: weight, height, waist circumference and neck
 circumference, body mass index in kg/m2, hours of fasting, systolic blood pressure, diastolic
 blood pressure and pulse pressure (mmHg). Blood pressure will be measured 3 times (Omron 6®)
 at 2-min intervals and the mean of the last 2 will be collected. Dried capillary blood
 testing (fingertip puncture) will be used to determine creatinine, uric acid and total
 cholesterol, using the reflotron® Plus system (Roche). In cases in which total cholesterol is
 greater than 200 mg/dl, the complete lipid profile (HDL cholesterol, LDL cholesterol and
 triglycerides) will be determined using Cobas B 101® system, Roche. Non-HDL cholesterol
 levels, glycated hemoglobin (Cobas B 101 system, Roche), the CKD-EPI glomerular filtration
 rate will be determined as well. Albuminuria and albumin/creatinine ratio will be determined
 in urine sample from spontaneous micturition collected in the MU using Clinitek Microalbumin
 2 Reagent Strips and a Siemens Clinitek Status® analyzer.

 ARTERY ULTRASOUND:

 The examination and reading will be performed by 2 nurses specialized in vascular imaging
 diagnostics, following a standardized protocol with the patient in supine decubitus. The
 VIVID I version BT12 ultrasound system (GE Healthcare) will be used. The system has a
 12L-RS/4-13 MHz linear probe (carotid and femoral arteries), a 4C-RS/1.5-6 MHz convex probe
 (abdominal aorta) and a 3SRS/ 1.5-2.5 MHz sector probe (transcranial). It also has a module
 for measuring intima-media thickness and pulsed Doppler ultrasound to assess hemodynamic
 abnormalities in the case that atheromatous plaques are present, and also to analyze the
 intracranial circulation. The DICOM network connectivity system will be used to record the
 results and ultrasound images online in the e-CAP.

 Carotid arteries: a total of 8 vascular regions will be analyzed (common carotid, bifurcation
 or bulb, internal carotid and external carotid), with the patient in supine decubitus and the
 head turned 45º towards the opposite side in the examination.

 Femoral arteries: with the patient in supine decubitus, the presence of plaque will be
 examined in the common femoral artery (1 cm proximal to the bifurcation) and the superficial
 femoral artery on both sides. The plaque definition follows the same previous criteria.

 Abdominal aorta: in men age 60 and older. With the patient in supine decubitus, the abdominal
 aorta will be examined in the midline of the abdomen from the base of the sternum until the
 bifurcation the iliac arteries. In an axial section, at the points where a greater diameter
 is observed, 2 images of the aorta will be captured, and 2 measurements will be made
 (anterior-posterior and lateral-lateral). An aortic aneurysm will be considered to be present
 when the diameter is greater than 3 cm.

 Transcranial ultrasound: the arteries of the Willis circle and their branches will be
 insonated through the transtemporal and transforaminal acoustic windows. The Doppler spectrum
 of each intracranial artery will be determined using the colour-coded signal. Flow direction,
 peak systolic velocity, mean flow velocity and diastolic flow velocity will be established.
 The intracranial carotid artery, the medial cerebral artery in the M1 and M2 segments, the
 anterior cerebral artery (segment A1) and the posterior cerebral artery in segments P1 and P2
 will be studied through the transtemporal acoustic window. The V4 segment of the vertebral
 arteries and the basilar artery will be studied through the transforaminal acoustic window.
 In each patient, the number, location and severity of stenosis will be recorded.
 Baumgartner's criteria will be used to establish the severity of the stenosis based on peak
 systolic wave velocity (moderate to serious stenosis, if it is ≥155/ 220 cm/s for the medial
 cerebral artery, ≤120/≥155 cm/s for the anterior cerebral artery, ≥100/≥145 cm/s for the
 posterior cerebral artery and the basilar artery, and ≥90/≥120 cm/s for the vertebral
 artery).

 ANKLE-BRACHIAL INDEX:

 A continuous Doppler (Hadecco ES100X MiniDop®), sphygmomanometer and blood pressure cuffs
 (Riester minimus III®) will be used. Systolic blood pressure will be measured in the brachial
 artery, posterior tibial artery and dorsalis pedis artery in both limbs. The ratios between
 tibial and pedal systolic blood pressure in each leg and the higher brachial blood pressure
 will be calculated. The final value for each limb will be the lower value of those obtained
 between tibial and pedal blood pressure. An ankle-brachial index value <0.9 will be
 considered to be suggestive of stenosis; a value <0.7 will be considered to be stenosis and a
 value ≥1.4 will be considered to be suggestive of arterial rigidity.

 SPIROMETRY:

 Spirometry will be used to assess lung capacity. It will be performed by the same nurse who
 will measure forced vital capacity (FVC); forced expiratory volume in one second (FEV1); the
 ratio between FEV1 and FVC and the lower limit of normality, as a percentage.

 DETERMINATION OF ADVANCED GLYCATION-END PRODUCTS:

 They will be measured using skin autofluorescence (SAF) in the forearm with the AGE Reader
 system® (Diagnostics, the Netherlands). SAF is measured using spectrophotometry which is
 calculated as the relationship of the intensity of reflected light compared to refracted
 light. The result obtained will be divided into 4 CV risk groups, taking into account age and
 sex.

 ATRIAL FIBRILLATION SCREENING:

 The presence of atrial fibrillation will be assessed with the SRA device® (EVINA Health
 Solutions) for one hour in the MU.

 PULSE WAVE VELOCITY:

 Pulse wave velocity (PWV) will be used as an indicator of arterial stiffness. It will be
 measured non-invasively with the carotid to femoral PWV (cfPWV) according to standard
 protocols.

 BIOLOGICAL SAMPLE COLLECTION:

 Blood samples from a peripheral vein of the hand or forearm to obtain serum, plasma, DNA and
 RNA will be collected. Moreover, a saliva sample will be collected.These samples will be
 prepared in aliquots following a standardized protocol, and sent frozen (dry ice) to a
 centralized biobank (IRBLleida Biobank, Spain) for processing and storage for subsequent
 studies of CV, inflammatory and mineral-metabolism biomarkers and genetic polymorphisms.
 Urine samples will be frozen and stored in a biobank for subsequent study of biomarkers.

 STATISTICAL ANALYSIS:

 Descriptive analysis will include absolute and relative frequencies for the qualitative
 variables, means and standard deviations for the continuous variables that follow a normal
 distribution, and median and interquartile range in the cases that do not follow a normal
 distribution. Their distribution will be analyzed with the chi-squared test, in the case of
 qualitative variables, and Student's t test or ANOVA for quantitative variables with a normal
 distribution, or, failing this, non-parametric Mann-Whitney U or Kruskal-Wallis tests will be
 used for those without a normal distribution. The correlation between quantitative variables
 will be analyzed using Pearson's or Spearman's test, depending on the distribution. The
 existence of collinearity between 2 variables will be analyzed to enter the variable that
 better predicts the result variable in the multivariate model. Multivariate analysis will be
 performed using logistic regression if the dependent variable is qualitative or linear
 regression if it is quantitative. Beta regression coefficients, odds ratios (ORs) and their
 respective 95% confidence intervals will be estimated as measures of association. In the
 longitudinal study, the incidence of CV morbidity and mortality and mortality due to any
 cause will be analyzed, based on the diagnostic tests used in the MU and the biomarkers
 studied using Cox regression. Statistical significance will be set at a p value < 0.05.
Inclusion Criteria:

 -  women (50-70 years) and men ( 45-65 years) with at least one cardiovascular risk
 factor (hypertension, dyslipidemia, obesity (BMI >30 Kg/m2), current smoking habit or
 former smoker (<10 years), first-degree family history of early cardiovascular
 disease).

 Exclusion Criteria:

 -  Prior medical history of cardiovascular disease.

 -  Diabetes.

 -  Glomerular filtration rate (CKD-EPI < 60 ml/min/1.73m2).

 -  Active neoplasm or acute disease.

 -  A life expectancy < 18 months.

 -  Pregnancy.