Study area and selection of participants
Participants were recruited from 19 villages within 50 kilometers radius of Komono (3°16’39”S, 13°13’14”E), a small town located approximately 60 kilometers from Sibiti, the capital of the Lékoumou Division in the Republic of Congo. This study site is located in a forested region where loiasis is endemic35.
The recruitment process was carried out in two phases. Mid-2023, a population survey was conducted to screen for loiasis in the villages near Komono. In June 2024, individuals between 18 and 70 years old, weighing between 40 and 90 kg and presenting with more than 3000 L. loa mf/mL during the first survey were invited for re-evaluation to determine their eligibility for the trial. This MFD threshold was arbitrarily set to ensure the individuals would still be microfilaremic at the start of the trial proper, given the relative stability of MFD over a one-year interval36.
Volunteers underwent a thorough medical evaluation. Participants were excluded if they had taken part in any non-observational studies or received vaccinations within four weeks prior to the study. Those with acute infections requiring treatment within the past ten days or with a history of neurological or neuropsychiatric disorders, including epilepsy, were also excluded. Individuals using medications known to interact with LEV, such as clozapine or phenothiazines, within ten days before the study, or those with known immunosuppressive conditions or a history of agranulocytosis, were not eligible. Additionally, participants who used cocaine or other illicit drugs within 72 h before treatment, had known intolerance to LEV, or had donated more than 500 mL of blood in the past eight weeks were excluded. Any condition deemed to pose an undue risk by the investigator also led to exclusion. Additional exclusions included women pregnant for fewer than three months, detected systematically with urinary rapid diagnostic test, and those who had received an anthelminthic in the month preceding the trial. All microfilaremic individuals during the 2023 survey and fulfilling these criteria in 2024 were eligible for inclusion. The clinical trial was conducted from July to September 2024.
Study design
For safety assessment, an independent Data Safety Monitoring Board reviewed the safety and efficacy results and could be consulted in the event of any unexpected clinical anomaly or SAE. Five days before treatment (D-5), all participants underwent a medical examination and completed a questionnaire to check for inclusion and exclusion criteria (see below). Once inclusion and non-inclusion criteria were met and the informed consent form was signed, participants were allocated to one of 3 arms: LEV 2.5 mg/kg during 3 days (LEV-3), LEV 2.5 mg/kg during 5 days (LEV-5), or placebo (PLA). Safety was checked until D30.
To assess efficacy, Loa MFD was measured just before the administration of the first tablet of treatment (D1), at day 3 (D3—48 h after the first dose), day 5 (D5—96 h after the first dose), at day 7 (D7), day 15 (D15), and at day 30 (D30) post-treatment. At D3, D5, and D7, each participant underwent a medical examination and screening for any adverse events (AEs). A medical team visited the villages of all participants every day from D2 to D7 to record and manage AEs. Conjunctivae were systematically examined, and if any abnormalities, febrile symptoms, or headaches were observed, a fundoscopic examination was conducted with a non-mydriatic retinal camera (Aurora, Optomed, DiTE sarl, Paris). All subjects received a participant card with emergency contact information.
Randomization, blinding and drug preparation and administration
A 1:1:1 randomization of 3 arms (LEV-3, LEV-5, or PLA). with blocks size of 3 was performed. Randomization was carried out by an independent statistician, with stratification based on sex, age and Loa MFD. For the study, sealed containers were prepared, each containing the appropriate number of LEV tablets—based on the participant’s weight—or matching placebo. Throughout the five-day treatment period, participants took their medication under the supervision of the trial’s physicians. Individuals randomized to the LEV-5 arm received 5 days of LEV, those in the LEV-3 arm received LEV from D1 to D3 then placebo from D4 to D5, and those in the PLA arm received placebo for 5 days. All procedures were double-blinded. All tablets were sourced from ACE Pharmaceuticals BV in Zeewolde, The Netherlands.
Laboratory procedures
Loa MFD were measured by examining two 50 μL calibrated blood smears (CBS1 and CBS2) on D1, D3, D5, D7, D15, and D30. To account for the diurnal fluctuation of Loa MFD36, blood samples for CBS preparation were collected between 10:00 AM and 3:00 PM. For consistency, smears from each participant were prepared at the same hour for each time point (mean difference with D1 ± 2.8 min; with 90% of our samples having a difference <26 min). Blood was obtained by finger-prick and spread onto two labeled slides, which were then dried at room temperature, dehemoglobinized, and stained with Giemsa within four hours. Two experienced technicians examined independently each slide under 100 × magnification. If the MFD readings differed by more than 35% between the two microscopists, the slides were re-examined blindly. The arithmetic means of the MFDs from the four readings (CBS1 by both readers and CBS2 by both readers) were calculated and expressed in mf/mL for the analyses.
Objectives and outcome measures
The primary objective of the trial was to evaluate the safety of multiple-dose LEV in individuals with Loa microfilaremia. The primary outcome measures were (i) the occurrence of an SAE and (ii) the frequency of AEs during the first month post-treatment.
The secondary objective was to assess the effect of LEV on L. loa MFD measured by: (i) the MFD reduction rates at D3, D5, D7, D15, and D30, (ii) the proportions of subjects with MFD reduction rates ≥40% and/or ≥80% at each time point post-D1, and (iii) the efficacy comparison between our arms. Reduction rates (%) were calculated as follows: ((MFD at D1)–(MFD at DX))/(MFD at D1) multiplied by 100 with X = 3, 5, 7, 15, or 30 days. Finally, we evaluated the proportion of individuals whose MFD fell below the thresholds of 30,000 mf/mL at D5 and D7.
Sample size calculation
Although our primary objective was safety, the absence of any data on SAE risk from our previous clinical trial13, and the fact that SAE risk in L. loa-microfilaremic individuals depends directly on the number of mf paralyzed and/or destroyed in the first 24–48 h post‑treatment, led us to base our sample‑size calculation on treatment efficacy. In that trial, 17.4% of participants who received a single 2.5 mg/kg dose of LEV showed a ≥40% reduction in their initial MFD two days after treatment, compared to 1.2% in the placebo group. Assuming an additive effect over a longer treatment period (with a 3-day treatment expected to be three times more effective than a single dose), we estimated that a 3-day treatment would reduce initial MFD by ≥40% in 52.2% of subjects, and a 5-day treatment would achieve this in 87.0% of subjects. Based on these estimates, we calculated that including 27 participants per treatment group would provide 80% power to detect these differences in MFD reduction. To account for a 20% loss to follow-up, we aimed to enroll a total of 99 subjects (33 per group).
Statistical analysis
Safety was assessed using the proportions of participants with at least one AE. Proportions were tabulated by AE severity score (CTCAE grading scale version 5.0, see Supplementary Information. Text 1) and arms, and compared using a two-sided exact Fisher test. Then, we performed logistic regression adjusted on L. loa MFD at D1 ( <20,000, 20,000–29,999, and >30,000 mf/mL—which are the same categories used in our efficacy analysis.), age, sex, treatment arm, and percentage of reduction of L. loa MFD at D3 ( <25%, 25–49%, and ≥50%).
For the efficacy analyses, L. loa MFD as well as reduction rates of L. loa MFD were calculated and compared between arms at D3, D5, D7, D15, and D30, using a two-sided non-parametric Kruskal-Wallis test following by an adjusted P values using Dunn test applying a Holm correction for multiple tests. The proportions of participants with MFD reduction exceeding 40% and 80% were compared between arms with two-sided Fisher’s exact tests at D3, D5, D7, D15, and D30, using a Holm correction for multiple tests. We first performed a modified intention-to-treat (mITT) analysis—our primary analysis—and secondly, a per-protocol (PP) analysis. We used a mixed-effects linear model to assess the percentage of pre-treatment L. loa MFD retained after treatment. This dependent variable was calculated as: (MFD at DX/MFD at D1), multiplied by 100 with X = 3, 5, 7, 15 or 30 days. This formulation represents the proportion of the initial microfilarial load that remained at each follow-up time point. The model included treatment group, time point, and their interaction as fixed effects, with adjustments for age, sex, and baseline MFD category ( <20,000, 20,000–29,999, and >30,000 mf/mL). Random intercepts and slopes for time points were specified for each participant to account for within-subject correlations, using an unstructured covariance matrix. The interaction between time points and treatment group was assessed with a likelihood ratio test. Baseline L. loa MFD was categorized because the linearity test was not significant, and this categorization was validated against the continuous variable using AIC and BIC. After fitting the model, we estimated our ICC. Finally, post hoc contrasts were performed to compare treatment efficacy between pairs of groups at each time point, assessing the significance of these differences (applying a Wald’s test). Finally, given the commonly used threshold of 30,000 mf/mL to determine when to initiate IVM treatment after an albendazole course, we reported the proportion of individuals with L. loa MFD >30,000 mf/mL before treatment who fell below this threshold at D5 and D7.
All statistical analyses were performed using Stata 18 (StatCorps LP, College Station, Texas, USA). The figures were created using R software (version 4.3.2).
Trial registration and ethic statement
This study was approved by the Committee on Ethics of the Foundation for Medical Research in Congo (No. 51/CEI/FCRM/2024). An Administrative Authorization (No. 000056/MSP/DGSSSa/DPM-19) was released by the Ministry of Health and Population of the Republic of the Congo. The French National Commission on Informatics and Liberty (CNIL DR-2024-099) approved that the study protocol was ensuring compliance with data protection regulations. This study was conducted in accordance with the rules of Good Clinical Practices. All participants signed an informed consent form before initiation of any study-related procedure. This trial is registered as number NCT06252961 in https://clinicaltrials.gov/.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
