Study design

This was a cross-sectional descriptive study conducted between May and June 2022 as part of the Morbidity due to Loiasis (MORLO) project which aims at assessing the morbidity and the prevalence of organ complications related to loiasis. Data were collected from participants recruited from 21 villages in the Lekoumou division of the Republic of Congo, a forest area endemic to loiasis with a combined population of 16,284 inhabitants (Census 2023).

Participants

Participants were recruited from a sample of volunteers aged 18 years and over who participated in a 2019 pre-screening survey for inclusion in a clinical trial for the treatment of loiasis²⁸. Individuals with a Loa loa microfilarial load exceeding 500 mf/ml in 2019 were matched with a control of the same sex and age (within 5 years) residing in the same village, and having no microfilariae at the pre-screening. A flow diagram of the pre-screening and selection process for this study was published in a previous article¹⁵.

Data collection

After identifying eligible participants in the database, they were invited to Sibiti Hospital where data was collected. Participants who consented to participate in the study underwent a clinical examination during which sociodemographic data and medical history were collected, including assessment of current medications, known cardiovascular or renal diseases, and other chronic conditions. Anthropometric measurements and blood pressure were taken. Stool examination was performed in participants who agreed to provided samples to screen for soil-transmitted helminths. Participants with fever or clinical symptoms suggestive of malaria were excluded. Whole blood samples were collected from each patient and analyzed for HbA1c and lipid profile with a point-of-care Afinion 2 device (Abbott Rapid Diagnostics, Bièvres, France). Additionally, a calibrated thick blood smear was performed following inclusion to quantify microfilarial density, as the 2019 screening was used solely to pre-identify potential participants. Finally, participants underwent an echocardiography to assess cardiac morphology and function.

Echocardiographic examination

Echocardiography was performed on all. Two-dimensional echocardiography with m-mode, pulse wave (PW), continuous wave (CW), and tissue doppler imaging (TDI) was performed with a CX50 ultrasound system (Philips). The acquisition protocol adhered to the standards of the American Society of Echocardiography and the European Association of Cardiovascular Imaging²². Echocardiographic examinations were performed by two qualified operators from the University Hospital of Montpellier: a certified cardiologist-sonographer (VD, 2/3 of examinations) and an anaesthesiologist from the cardio-surgical intensive care unit (LR, 1/3 of examinations). To ensure data consistency, all final interpretations and measurements were centralized and conducted exclusively by the cardiologist-sonographer following a standardized quality control process.

Echocardiographic measurements

A comprehensive echocardiographic evaluation was performed to assess ventricular and atrial size, left and right ventricular systolic and diastolic function, regional wall motion abnormalities, valvular structures and function, and evaluation of pericardium adhering to the latest international guidelines and recommendations^22,29. Image quality was excellent for the vast majority of participants, with only a very small number of individuals having suboptimal echogenicity that did not compromise satisfactory echocardiographic evaluation. Left ventricular (LV) end-systolic and end-diastolic volumes, as well as LV ejection fraction (EF), were calculated using the biplane method of disks (modified Simpson’s rule).

LV diastolic function and left atrial filling pressure (LAP) were evaluated using mitral inflow velocities (E/A ratio), septal mitral annulus velocity (e’), tricuspid regurgitation (TR) velocity, E/e’ ratio and left atrial (LA) volume index³⁰.

Right ventricular (RV) function was assessed using tricuspid annular plane systolic excursion (TAPSE), systolic excursion velocity of the tricuspid annulus by tissue Doppler imaging (s’), and fractional area change (FAC). Pulmonary artery pressure was estimated using TR velocity. Right atrial pressure was estimated based on the diameter of the inferior vena cava (IVC) and its inspiratory collapse^22,31.

The following variables were indexed to BSA for analysis: LV septal wall thickness, LV posterior wall thickness, LV end-diastolic volume (2-chamber and 4-chamber), LV end-systolic volume (2-chamber and 4-chamber), LV mass, LA area (2-chamber and 4-chamber), LA volume, RV basal diameter, right ventricular outflow tract (RVOT) prox, RV end-diastolic area, RV end-systolic area, RA area, aortic sinotubular junction diameter, sinus of Valsalva diameter, ascending aorta diameter, Cardiac output, and aortic valve area. Indexing to BSA was performed to account for variations in body size among the study population. All echocardiographic examinations were systematically reviewed and validated by an experienced cardiologist-sonographer to ensure consistency and reliability of measurements.

Construction of reference values

For the construction of reference values charts, we considered the group of healthy participants as those with a BMI between 18 and 35 kg/m², without hypertension (blood pressure ≥140/90 mmHg or a history of hypertension under treatment) or diabetes (HbA1c > 6.5% or known diabetes under treatment), and without any significant morphological abnormalities on echocardiography. Specifically, we excluded individuals with significant valvulopathy (mitral or aortic insufficiency of grade 3 or 4, tricuspid insufficiency of grade 3 or 4), pulmonary stenosis, dilated or hypertrophic cardiomyopathy, suspected cardiac amyloidosis, regional wall motion abnormalities (hypokinesia, dyskinesia), restrictive filling patterns, significant pericardial effusion, or congenital anomalies such as severe ventricular septal defect or atrial septal defect. To account for the potential impact of chronic kidney disease on cardiac measurements, we performed a sensitivity analysis by additionally excluding participants with stage 3–5 chronic kidney disease using EKFC KDIGO classification. Echocardiographic measurements for each population age and gender subgroups were described using the mean and standard deviation. Reference intervals were determined using a parametric approach, calculated as the mean ± 2 standard deviations (SD), applied to the healthy population.

Diagnostic of cardiac abnormalities

Once these reference charts were established, we aimed to classify our entire population according to ten distinct cardiac abnormalities that were defined based on the reference values derived from the healthy population examined as part of the present study, but also from reference values obtained from American and European populations (Table 5 and Supplementary Tables). To minimize the total number of comparison articles and consequently reduce variability in methodological approaches, we prioritized studies with the maximum number of available echocardiographic parameters. External reference values were primarily derived from the NORRE study. Additional reference standards were obtained from established international guidelines: British Society of Echocardiography recommendations, American Society of Echocardiography and European Association of Cardiovascular Imaging joint recommendations and European Association of Cardiovascular Imaging and American Society of Echocardiography guidelines^{7,19,20,21,22}. Table 5 presents these ten abnormalities and definitions. Last, impaired diastolic function was assessed according to the recommendations of the European Association of Cardiovascular Imaging (Supplementary Fig. 1)³⁰. As TR velocity was exclusively evaluated in participants presenting with TR, individuals without this condition were assumed to have a TR velocity

Statistical analysis

Quantitative variables were described using mean ± standard deviation, median (and interquartile range), and range. Categorical variables were described using frequency and percentage. For the identified morphological anomalies, we described the distribution by sex and age group. The chi-squared test or Fisher’s exact test was used to compare proportions, depending on whether the expected cell count was less than or equal to 5. For echocardiographic measurements, a parametric Student t-test was used to compare values between sexes, and ANOVA test was used to compare values between age groups. P values were adjusted for multiple comparisons using the Holm correction method. Once this work of constructing reference charts for echocardiographic norms was completed, we applied these cut-offs to classify individuals based on 10 clinically significant echocardiographic abnormalities (Table 5). Data were initially collected on paper forms in the field and subsequently entered into a REDCap (Research Electronic Data Capture) database by two independent operators, with discrepancies resolved by a third operator to ensure data accuracy. Data were analysed using R version 4.2.3 (2023-03-15 ucrt) and RStudio version 2023.6.1.524 (Integrated Development Environment for R. Posit Software, PBC, Boston, MA.) with tidyverse and gtsummary packages^32,33.

Inclusion and ethics statement

Ethical approval for this study was granted by the Ethics Committee of the Congolese Foundation for Medical Research (No. 036/CIE/FCRM/2022) and administrative authorization by the Congolese Ministry of Health and Population (No. 376/MSP/CAB/UCPP-21). All participants provided written informed consent after receiving a comprehensive explanation of the study. The study was conducted in accordance with the fundamental principles of ethics in research involving human subjects, as outlined in the Declaration of Helsinki³⁴.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.