Disease activity index is associated with subclinical atherosclerosis in childhood-onset systemic lupus erythematosus

This cross sectional study was performed between April 2017 and May 2018. cSLE patients followed in the Paediatric Rheumatology Clinic of the Clinical Hospital of Ribeirão Preto Medical School, Brazil, a tertiary referral center, were invited to participate. All patients fulfilled the American College of Rheumatology classification criteria for SLE [9], had disease onset at less than age 18 years, and were recruited up to 21 years of age. Pregnant individuals and patients with infection were excluded.

Clinical data, such as demographics, lupus-related clinical history, comorbidities, traditional risk factors for atherosclerosis, including hypertension, dyslipidemia, obesity, diabetes and use of contraceptives were retrieved from the patient’s medical records. Current and previous medication treatment - prednisone, intravenous methylprednisolone pulse, hydroxychloroquine sulphate, methotrexate, azathioprine, cyclosporine, mycophenolate and intravenous cyclophosphamide - were also recorded.

The prescribed corticoid was evaluated in total actual prednisone equivalent dose (mg), accumulated dose in grams, and in average dose by kg/day during the patient treatment. The patients were stratified into three groups based on the average dose of corticoid used for disease treatment: low dose, < 0.15 mg/ kg/day; moderate dose, 0.15–0.40 mg/ kg/day; high dose, > 0.40 mg/kg/day. The categorization of the corticosteroid dose was based on the relationship between the weight-adjusted prednisone dose and the CIMT measurement identified in the APPLE study [6].

The Systemic Lupus Erythematosus Disease Activity Index - SLEDAI-2 K were used to assess disease activity and to classify the patients by activity level: 0 no activity, 0–5 mild activity and, greater than 5 moderate to severe [10].

The Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI) - SLICC/ACR DI were used to assess disease activity and cumulative damage [11].

Physical examination of all patients was performed by the same pediatric rheumatologist (PBSM). Tanner’s pubertal staging was assessed to classify the patients into pre-puberty and puberty (Tanner stage ≥2) since that puberty may be related to cSLE disease activity [12, 13]. Hypertension was defined as systolic and/or diastolic blood pressure  95th percentile for children between 1 and 13 years and ≥ 130/80 mmHg for children ≥13 years [14].

Data on diet, smoking and physical activity were collected during the physical evaluation. Regular physical activity was considered when the patient performed 60 min or more of daily physical activity, with periods of vigorous activity performed at least 3 days per week, for the last 3 months [15].

Twelve-hour fasting exams included complete blood cell count, complement levels (C3 and C4), anti-dsDNA, homocysteine, high sensitivity C-reactive protein (hsCRP), cyanocobalamin, folate, total cholesterol, triglycerides, high density lipoprotein, low density lipoprotein, glucose levels, urine analysis and spot urine protein creatinine ratio. Cut-off values were determined based on the assay manufacturer’s instructions.

Proteinuria was defined as protein to creatinine ratio > 0.2 in spot urine sample and moderate to severe proteinuria for ratio ≥ 1 [16]. Creatinine clearance (CrCl) was calculated using the Schwartz formula [17].

Plasma levels of interleukins (IL), IL-1alpha and IL-1beta, IL-6, IL-10, IL-17; interferon (IFN) alpha and beta and adhesion molecules, vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and P-selectin were measured using commercial Kits - Human ELISA kits - R&D Systems Inc.® (Minneapolis, MN, USA).

The anthropometric measurements were weight, height, body mass index (BMI) [18] and waist circumference (WC) [19], measured according to published methods. Electrical bioimpedance analysis was performed and the percentage of body fat was determined according to McCarthy, 2006 [20].

Food intake was assessed by one 24-h dietary recall (24 h-R) applied to participants on the same day of physical exam and blood sample collection [21]. A picture booklet of food portion size (small, medium and large) was used to convert the food to g or ml [22]. Energy, macronutrients and micronutrients were calculated using the software specially developed to analyze the dietary intake of the Brazilian population (Dietwin® version 1997–2002).

The Healthy Eating Index (HEI) was calculated using data obtained from the 24 h-R. The HEI considered in this study was previously revised for the Brazilian population [23] and subsequently validated for children and adolescents [24]. It consists of an indicator that simultaneously analyzes several nutritional components, based on energy density, evaluating their quality, regardless of the amount of food consumed, represented by a single value that can be classified as: “poor diet”, scores less than 65; “good diet”, scores ≥85; and two intermediate categories, 65–74 and 75–84, “need adaptation in the diet” [24]. The maximum index score is 100.

To determine the prevalence of subclinical atherosclerosis an expert radiologist (SRT) performed US to assess the CIMT using a Logic E9 device (General Electric Company) with high frequency linear transducers (ML6–15 MHz) and following international standardized protocols [7]. US was performed in a controlled room temperature at 23 °C, in the morning (between 7:30 am and 9:00 am), with the patient resting comfortably for 15 min prior to acquiring data in the supine position. The patient’s neck was slightly extended and the head turned 45° towards the side opposite of that being examined. CIMT was automatically measured on longitudinal B-mode images using the Auto IMT software. After identifying the carotid bifurcation, CIMT was measured from three arterial segments three times each, as follows: the common carotid artery (CCA) from 10 mm proximal to the tip of the flow divider (TFD), the carotid bifurcation (from the TFD to 10 mm proximal to the TFD) and the proximal 10 mm of the internal carotid artery (ICA). The average of the three measures of each segment was used to compose data. CIMT measurements were also compared to reference values of a population study for children under 18 years old [25, 26] and for teenagers between 18 and 21 years old [27, 28]. CIMT greater than or equal to the 90th percentile for age and sex was considered subclinical atherosclerosis [29] (Fig. 1).

The characteristics of the study sample were summarized using descriptive statistics with ordinal data presented as percentages and continuous data as means, standard deviations [SD] or medians and interquartile ranges, as appropriate. The study sample was divided into two groups, with and without subclinical atherosclerosis, according to the results of the CIMT measurements. Fisher’s exact test and Wilcoxon-Mann Whitney test were used to investigate associations between categorical and continuous variables with groups, respectively. Fitted log-binomial regression models were used to estimate prevalence ratios and their 95% confidence intervals. Linear regression models were adjusted to associate potential risk factors with CIMT. Statistical and clinical criteria were considered for selecting variables for multiple modeling.

Multiple correlation between continuous variables and CIMT measurements was assessed with Spearman’s rank order correlation coefficient. Both the absolute values and normalized percentiles of the CIMT measurements were analyzed. For statistical purposes, the CIMT values of the right and left carotids were averaged.

The SLEDAI-2 K was used as a continuous variable or as a dichotomous variable: SLEDAI-2 k > 5 score (yes or no), for considering SLEDAI values greater than 5 moderate to severe disease activity. The SLICC/ACR DI scores were included in the statistical analysis as a dichotomous variable with subjects grouped as score of 0 (no damage) or score ≥ 1 (damage present) [30].