https://orcid.org/0000-0001-5721-0891
Figures
Abstract
Background
Onchocerciasis, also known as river blindness, remains a public health challenge in tropical regions. Despite significant gains through mass drug administration (MDA) with ivermectin, the infection remains persistent in many endemic foci. While the disease burden has reduced, elimination may require alternative strategies, including macrofilaricidal therapies. Rifampicin (RIF) has demonstrated strong anti-Wolbachia activity in vitro and in animal models, but its clinical efficacy in humans remains uncertain.
Methods
We conducted a phase II randomised, controlled trial in Ghana to evaluate the safety and efficacy of high-dose RIF (35 mg/kg/day) combined with albendazole (ALB, 400 mg/day) as an alternative treatment for onchocerciasis. The study randomised 120 infected participants into one of four arms: RIF + ALB for 14 days (TA1), RIF + ALB for 7 days (TA2), ALB alone for 14 days (TA3), and a No Treatment control group (TA4). Treatment efficacy, including Wolbachia and microfilariae (MF) depletion, adult worm vitality and embryogenesis were assessed following treatment at 4-, 18-, and 20-months.
Results
High-dose RIF + ALB was safe and well-tolerated, associated with only mild, transient adverse events. Treatment with RIF + ALB for 14 days reduced Wolbachia load in skin MF at 4 months (47.9%; p = 0.018). ALB monotherapy showed sustained reductions in skin microfilarial densities at the 18- and 20-month time points (p < 0.05). However, no macrofilaricidal effect or sustained Wolbachia depletion in adult worms was observed in any group.
Conclusion
High-dose RIF + ALB is safe with limited macrofilaricidal efficacy in clinical settings, contrasting with the superiority observed from preclinical trials. ALB alone shows promise as an alternative microfilaricidal treatment to ivermectin or as a complementary regimen. The lack of sustained efficacy of RIF as an anti-Wolbachia regimen may reflect suboptimal dosing, highlighting the need for further optimisation of RIF-based regimens for onchocerciasis treatment.
Trial registration
PACTR202009704006025 (https://pactr.samrc.ac.za/Search.aspx). Date of trial registration: September 9, 2020
Author summary
Onchocerciasis, commonly referred to as river blindness, is a disease caused by worms transmitted by blackflies. The disease mainly affects people living in the rural tropics, causing severe itching, skin changes, and vision loss. The main strategy for controlling the disease is through mass drug administration (MDA) with ivermectin, targeting the juvenile worms, with little or no effect on the adult worms. To complement elimination efforts, alternative drugs that can be administered within a short duration and able to kill the adult worms are urgently needed. In this study, we tested whether a combination of high-dose rifampicin (an antibiotic) and albendazole (an anti-worm medicine) could provide a better treatment option for onchocerciasis patients. We conducted a clinical trial in Ghana, enrolling 120 people with river blindness, and randomly assigning them to receive either high-dose rifampicin plus albendazole for 7 or 14 days, albendazole alone for 14 days, or no treatment. We followed participants for 20 months to assess the drugs’ safety and effectiveness in killing the juvenile and adult worms. We found that the combination of high-dose rifampicin and albendazole was safe and well tolerated, with only mild and temporary side effects. At 4 months after treatment onset, the 14-day combination treatment reduced the essential Wolbachia bacteria found in the juvenile worms, resident in the skin, but this effect was not observed in adult worms when assessed at 20 months. Treatment with albendazole alone also showed a reduction in the number of juvenile worms present in the skin. Regardless, none of the treatments had lasting effects that resulted in killing a significant number of the adult worms. Our results show that high-dose rifampicin plus albendazole is safe, but it does not effectively kill the adult worms or achieve long-term depletion of Wolbachia found in the worms. These findings highlight the need for further research to develop more effective treatments that could support the global goal of eliminating river blindness.
Citation: Ahiadorme M, Klarmann-Schulz U, Dubben B, Nadal J, Korir PJ, Pfarr K, et al. (2026) Assessing the efficacy of high-dose rifampicin plus albendazole against onchocerciasis. PLoS Negl Trop Dis 20(8): e0014594. https://doi.org/10.1371/journal.pntd.0014594
Editor: Benjamin L. Makepeace, University of Liverpool, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND
Received: March 1, 2026; Accepted: July 20, 2026; Published: August 12, 2026
Copyright: © 2026 Ahiadorme et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The data supporting the findings of this study are available in the institutional repository of the Kwame Nkrumah University of Science and Technology (KNUST) at the following link: https://ir.knust.edu.gh/items/84db2810-4e66-4e48-9783-3ef79373dc59. Use of these data requires proper acknowledgment of the source and appropriate credit to the original authors.
Funding: This research was funded by the European and Developing Countries Clinical Trials Partnership (EDCTP 2) under grant TMA2018SF-2451, awarded to AYD, and by Bundesministerium für Bildung und Forschung the German Federal Ministry of Education and Research (Bundesministerium fur Bildung und Forschung -BMBF) under grant 81204851, awarded to AYD. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Onchocerciasis, or river blindness, is a parasitic disease caused by the filarial nematode Onchocerca volvulus. It remains a significant public health burden in sub-Saharan Africa, with an estimated 20.9 million people infected globally, over 99% of whom live in Africa [1,2]. The disease leads to chronic skin conditions and irreversible blindness, causing substantial social and economic hardships in affected communities [3].
Ivermectin (IVM) has been the cornerstone of onchocerciasis control and elimination through annual or biannual mass drug administration (MDA) programmes in endemic areas [4]. It effectively reduces skin microfilariae (MF) loads and transmission potential, but does not kill adult worms, necessitating repeated treatments for over a decade [4]. Although IVM-MDA has led to the elimination of onchocerciasis in some regions such as Mali and Senegal [5,6], progress remains slow in areas with intense transmission and high parasite burdens [7–9]. Moreover, its use in Loa loa co-endemic areas can cause severe adverse events (SAEs), including encephalopathy and death due to rapid L. loa MF destruction [10,11].
An extensively explored alternative is anti-Wolbachia therapy which targets Wolbachia endosymbiotic bacteria essential for the survival and reproduction of many filarial parasites, including O. volvulus [12,13]. Antibiotic-mediated depletion of Wolbachia, disrupts embryogenesis, suppresses MF production, and ultimately kills adult worms [14–16]. This macrofilaricidal activity arises from the crucial role Wolbachia plays in germline function; depletion of Wolbachia triggers widespread apoptosis in reproductive tissues, thereby blocking embryogenesis and sterilising adult worms [17]. A 4–6-week administration of doxycycline (DOX; 100–200mg/day) in onchocerciasis has shown evidence of these effects. However, this requires a prolonged treatment duration and has been shown to be contraindicated in pregnant women and children under 8 years of age, posing logistical and ethical challenges for large-scale implementation [18–21].
Recent preclinical studies have identified rifampicin (RIF), a widely used antibiotic for tuberculosis, as a potent anti-Wolbachia agent. A compelling rationale for combining RIF with albendazole (ALB) lies in ALB’s dual activity against both the filarial nematode and potentially, its Wolbachia endosymbiont. ALB is a well-established anthelmintic that disrupts nematode microtubules via β-tubulin inhibition. In addition, earlier cell-based and molecular studies demonstrated that its metabolite, albendazole sulfone (ALB-SO2), can directly reduce Wolbachia titres by interfering with FtsZ-mediated bacterial cell division [22,23]. Since FtsZ is encoded by Wolbachia and plays an essential role in cytokinesis, it represents an attractive drug target for antifilarial therapy [24]. However, in a study by Turner et al. (2017), administration of ALB alone had no effect on Wolbachia depletion, but when combined with RIF, it significantly enhances bacterial clearance [25]. Specifically, a 7-day RIF plus ALB regimen achieved >99% Wolbachia depletion in a Brugia malayi mouse model, with partial but significant reductions in female worm burden [25]. While a minimum of 14 days of high-dose RIF was required to achieve >90% Wolbachia depletion in male O. ochengi implants in an SCID murine model [12], it is worth noting that male O. ochengi worms are considerably smaller in biomass than adult O. volvulus worms, which may limit direct extrapolation of these findings to the clinical setting. These findings, nonetheless, provided the preclinical basis for the 7- and 14-day treatment durations evaluated in this current trial. Importantly, RIF at high-dose of 30–40 mg/kg is bioequivalent to the exposures used in animal models, and has been established to have favourable safety profiles in humans, including in children and pregnant women [26]. This combination therapy could potentially address the limitations of IVM and DOX, and provide a safer, more practical MDA regimen capable of achieving macrofilaricidal activity within a feasible timeframe.
Given these promising findings, it is crucial to evaluate the efficacy and safety of high-dose RIF plus ALB in human clinical trials. It is important to determine whether the combination therapy effectively kills or sterilises adult O. volvulus worms, suppresses or clears skin MF, and depletes Wolbachia in both MF and adult worms. Such evidence could provide a solid basis for the adoption of this regimen as a new tool to complement onchocerciasis elimination efforts, especially in areas where existing MDA strategies are inadequate or unsafe. This study sought to address these knowledge gaps by assessing the efficacy of high-dose RIF (35 mg/kg/day) combined with ALB (400 mg/day), administered for 7 or 14 days in a phase II randomised controlled clinical trial. The study evaluated the macrofilaricidal efficacy of different treatment regimens on adult O. volvulus worms, impact on embryogenesis in adult female worms, and effect on microfilaridermia. The study also assessed the effectiveness of the regimens in depleting Wolbachia endobacteria, using qPCR in skin MF and immunohistology techniques. In addition, the safety profile of the combination therapy was evaluated.
Materials and methods
Ethics statement
The study protocol, informed consent and case report forms received ethical approval from the Committee on Human Research, Publication and Ethics (CHRPE) of the School of Medical Sciences (SMS) of the Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana, the Ghana Health Service (GHS) Ethics Review Committee, and the Ghana Food and Drugs Authority (FDA).
Study population
The study was conducted in the Sefwi Akotombra district in the Western North Region of Ghana, a predominantly rural and agriculturally driven district recognised as an endemic district for onchocerciasis. The district is characterised by the Tano river and its tributaries (Yoyo, Kunuma, Suhien, and Sui), which serve as major water sources, supporting both agriculture and the local ecosystem [27]. Twenty-five (25) accessible communities were purposively selected for the trial, reflecting the distribution of the disease in the district.
Onchocerciasis control in the Sefwi Akotombra district has relied on annual mass drug administration (MDA) with ivermectin (IVM) since 1994, coordinated by the Ghana National Neglected Tropical Disease (NTD) Control Programme, and delivered through Community Drug Distributors (CDDs) or Community Health Volunteers (CHVs). In 2018, biannual MDA was introduced to intensify control efforts. However, no MDA activities were conducted in 2020 due to the COVID-19 pandemic, in accordance with WHO recommendations [28,29].
The trial was registered in the Pan African Clinical Trials Registry (PACTR202009704006025) and conducted in compliance with the Declaration of Helsinki (2013 revision) and Good Clinical Practice (GCP) guidelines. Oversight was provided by a Trial Steering Committee (TSC) and an independent Data Safety and Monitoring Board (DSMB).
Eligible participants aged 18–55 years, weighed at least 40 kg, had at least one palpable onchocercoma with confirmed positive skin MF, and were in good general health. Participants were excluded based on a history of tuberculosis (TB), intolerance to the study medications, pregnancy, breastfeeding, substance or alcohol abuse, abnormal renal and hepatic enzymes, glucosuria, hypertension, or any condition requiring long term medication.
Written informed consent was obtained from all participants, either by signature or thumbprint. Participation was voluntary, and individuals retained the right to withdraw from the study at any given time.
Study design, sample size, randomisation and intervention
This study was a non-confirmatory randomised, open-label, phase II pilot trial, designed to evaluate the efficacy and safety of high-dose RIF combined with ALB for the treatment of onchocerciasis. As a pilot study, the sample size was determined based on precedent from similar studies, rather than on statistical argumentation. According to Julious (2005) [30], a minimum of 12 participants per treatment arm is recommended for pilot trials to provide preliminary estimates of treatment effects and variability, while considering feasibility and regulatory guidance.
The study enrolled and randomised 120 participants into four treatment arms (30 per arm), accounting for a 30% dropout rate, consistent with previous onchocerciasis clinical trials. Treatments were administered under directly observed therapy (DOT) by the trial clinician and pharmacist within participants’ communities, beginning March 8, 2021. RIF and ALB were administered simultaneously, and participants were encouraged to avoid alcohol intake within the treatment period and to eat prior to taking the study medications to improve gastrointestinal tolerance, support treatment adherence, and ensure optimum absorption [31–33]. The treatment duration corresponded to the assigned arm, with a maximum of 21 days allowed to accommodate any missed doses.
The treatment arms were as follows:
- Treatment Arm 1 (TA1): Rifampicin 35 mg/kg/day plus albendazole 400 mg/day for 14 days
- Treatment Arm 2 (TA2): Rifampicin 35 mg/kg/day plus albendazole 400 mg/day for 7 days
- Treatment Arm 3 (TA3): Albendazole 400 mg/day for 14 days
- Treatment Arm 4 (TA4): No treatment (control)
The RIF-based regimens were informed by prior evidence supporting short-course, high-dose therapy [12,25] with ALB added to enhance antifilarial activity and to assess the feasibility of a shorter-course treatment approach [25,34]. RIF monotherapy was not included, as evidence indicates superior efficacy with combination regimens [34–36], and the additional arms were not feasible due to logistical and ethical constraints. An ALB monotherapy arm was included to reaffirm its potential role in onchocerciasis treatment, supported by findings that standard-dose ALB produces sustained reductions in microfilarial densities [37,38].
Participants and the study team were not blinded to the treatment allocations. However, outcome assessors conducting histological and qPCR analyses were blinded to minimise assessment bias.
All participants were actively monitored and assessed by the trial clinician during and after administration of study medications for the occurrence of adverse events (AEs). All study procedures were conducted in accordance with GCP guidelines to ensure the safety and rights of participants were safeguarded throughout the study period. Participants were followed up at 4-, 18- and 20-months post-treatment to assess treatment efficacy. IVM was administered, 6 months following treatment with RIF and ALB to all groups, including the no treatment arm. This was done in accordance with ethical considerations and standard of care.
Study outcome measures
The primary outcome of the trial was the proportion of viable adult O. volvulus worms in accessible nodules assessed by histology at 20 months post-treatment. Secondary outcomes included the assessment of embryogenesis within female adult worms, presence of free MF within nodules by histology at 20 months post-treatment, absence of Wolbachia endobacteria in skin MFs assessed by qPCR at 4-months post-treatment, qualitative and quantitative evaluation of Wolbachia depletion in adult worms at 20 months post-treatment using immunohistology and qPCR, respectively, and the presence or absence of skin MFs, detected by skin snip microscopy.
Parasitological assessment
All enrolled participants presented with at least one palpable nodule (onchocercoma). Skin MF loads were assessed using the standardised skin snip protocol, in which two bloodless 2-mm skin biopsies were taken from the left and right iliac crests and incubated overnight at room temperature in 1 mL of sterile physiological saline to allow MF emergence [39]. MF density was calculated as the arithmetic mean number of MF per mg skin snip. Two independent microscopists, blinded to treatment allocations, performed MF enumeration, and the mean of their counts was used for analysis [39].
Nodulectomy and histological assessment
At 20 months post-treatment, palpable nodules were surgically removed from consenting participants at the Sefwi Wiawso Government Hospital under aseptic conditions and local anaesthesia. Each nodule was labelled with participant identifiers, fixed in 80% ethanol, and processed using an STP-120 automated tissue processor (Microm International GmbH, Thermo Fisher Scientific, Walldorf, Germany) before embedding in paraffin wax. Serial sections of 5 µm were cut from the paraffin blocks using a pfm Rotary 3005E microtome (pfm medical gmbh, Cologne, Germany) for histological, immunohistological, and molecular analyses following established protocols.
A minimum of 8 sections per nodules were assessed by two experienced parasitologists blinded to the treatment allocations. For histology, sections were stained with Haematoxylin and Eosin (H&E; Merck, Darmstadt, Germany) to evaluate worm morphology including sex differentiation, embryogenesis, and the presence of MFs within nodular tissue. Iron deposits in worm tissues were detected using the Pearls Prussian Blue method with potassium ferrocyanide-hydrochloric acid solution and nuclear fast red counterstain [40], where the intensity of blue staining of the worm gut correlated with worm age: strong staining indicated older worms, light staining for younger worms, and absence of staining suggested dead worms. Worms lacking conclusive iron staining or those decalcified with EDTA were classified as not judgeable.
For immunohistological detection of worm vitality and Wolbachia endobacteria, the Dako REAL Detection System (Alkaline Phosphatase/RED, Labelled Streptavidin-Biotin (LSAB) method; Dako Cytomation, Hamburg, Germany) was employed. A rabbit anti-serum against Dirofilaria immitis Wolbachia surface protein (DiWsp) at a dilution of 1:2000 with REAL antibody diluent was used to detect Wolbachia [41]. Secondary detection used a REAL Biotinylated Secondary Antibody (AB2), followed by REAL Streptavidin Alkaline Phosphate (AP) labelling, with REAL Chromogen Red as the chromogen, and REAL Haematoxylin as counterstain. Additionally, a rabbit anti-serum against a cathepsin D-like lysosomal aspartic protease of O. volvulus (APR) at 1:2000 dilution with REAL antibody diluent was employed to assess worm vitality and estimate age: absence of APR staining, calcification, loss of cuticle, or organ degeneration indicated dead worms [42], while strong APR positivity in hypodermis indicated living worms [43,44], with staining intensity further used to classify age as newly acquired, or old [45]. Moribund worms with extensive degeneration but APR-positivity were classified as dead, especially if associated with neoplasms. Stained slides were digitalised at 20X magnification using a Zeiss AxioScan (Carl Zeiss, Jena, Germany) and analysed with ZEN 3.10 software (blue edition).
Wolbachia content was classified as ‘none’ (no visible staining), ‘few’ (less than 50 bacteria per hypodermis) and ‘many’ (densely packed bacteria), applicable to both adult worms and embryos [45]. Embryogenesis was evaluated by identifying developmental stages from oocytes to stretched MF in the uterus; normal embryogenesis required the presence of all stages [46,47].
DNA extraction from microfilariae and nodule sections
Genomic DNA was extracted from skin MF and paraffin-embedded nodule sections using the QIAamp DNA Micro Kit (50) [48,49], following the protocol described by Schlabe et al. (2022) [50]. For nodule samples, 8 sections (5 µm thickness) per nodule were processed. The same extraction procedure was used, except that nodule sections underwent an overnight proteinase K digestion at 56 °C to ensure complete tissue lysis. DNA was eluted in AE buffer and stored at -20 °C until further analysis.
Duplex qPCR assay
Quantification of Wolbachia-specific DNA in samples extracted from skin MFs and nodule sections was performed using a duplex qPCR assay targeting the Wolbachia single-copy gene ftsZ (wOvftsZ; GenBank: AJ276501) and the O. volvulus housekeeping gene actin (OvActin; GenBank: M84916.1), following the protocol described by Schlabe et al. (2022) [50]. The primer and probe sequences used in the assay are provided in S1 Table. The assay was run on a RotorGene 6000 (Qiagen, Hilden, Germany). Wolbachia copy number was normalised per MF (wOvFtsZ copy number/μL x elution volume ÷ number of MF) or relative to OvActin in nodule sections (wOvFtsZ copy number/μL ÷ OvActin copy number/μL) to account for variability in worm material across samples. Positive controls from our previous clinical trial (MoRion, ISRCTN43697583) were included in all assays runs to monitor qPCR performance and reproducibility.
Statistics
All statistical analyses were performed using Stata version 16 (Stata Corporation LLC, College Station, USA) and SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). Data visualisation was performed using GraphPad Prism 10.2 (GraphPad Software, Inc., San Diego, CA).
Continuous variables were assessed for normality using the Shapiro-Wilk test. Unless where stated, non-parametric tests were used for analysis. Analysis of baseline characteristics of participants included the Fisher’s exact test for sex and IVM rounds (grouped); Kruskal-Wallis test for number of IVM rounds, nodule counts, nodule sites and MFs/mg skin; and analysis of variance (ANOVA) for age, body weight and years lived in an endemic area.
In analysing treatment efficacy, three datasets were utilised:
- Per protocol (PP): Participants who completed treatment per the protocol.
- Intention-to-treat (ITT): Participants who took the drugs at least once.
- MF-PP: Skin snipped participants who were treated PP, and present at 4-, 18- and 20-months post-treatment and had taken IVM at the 6-months follow-up.
Given that the study was a non-confirmatory pilot trial, efficacy analysis primarily relied on the PP dataset. The ITT dataset was used to describe baseline characteristics, assess adverse events, and corroborate findings from the PP analyses. For nodulectomy-based histological outcomes, all individuals who underwent the procedure were included in the ITT analysis, without imputation for missing values.
Histological evaluation endpoints were analysed using alternating logistic regression, implemented in SAS-Procedure Genmod to account for potential dependencies among worms within a patient. The Odds Ratios (ORs) with 95% confidence intervals (CIs) were derived from regression models to assess differences between treatment groups regarding outcome measurements (occurrence of dead worms, Wolbachia depletion, wOvFtsZ, OvActin, wOvFtsZ/OvActin ratio, inhibition of embryogenesis).
In comparing treatment groups for MF-PCR, data analysis was performed using the Kruskal-Wallis test, followed by Dunn’s post hoc test for multiple comparisons, with the Holm-Bonferroni correction applied.
Within treatment (before and after treatment) differences in microfilarial loads (MF/mg skin snip) were assessed using the Wilcoxon-signed-rank-test. A p-value < 0.05 was considered significant.
Results
Participants flow and recruitment
A total of 120 participants were enrolled from 25 villages in the Sefwi Akontombra district and randomised 30 per treatment arm into 4 groups as detailed in the study flow chart (Fig 1). All participants received one of the four assigned treatments, and underwent follow-up evaluations at 4-, 18- and 20 months post-treatment. Participant recruitment and enrolment occurred between February 10 and March 8, 2021. Treatment was administered from March 8 to April 4, 2021, and follow-up was completed on December 7, 2022.
*Mentally ill (N = 1), Refused (consent withdrawn) (N = 3), Absence of medium-sized nodule (N = 1), Age > 55yrs (N = 1). **Pregnancy (N = 1), High GGT only (N = 8), High GGT and Epileptic (N = 1), eGFR < 60 only (N = 3), eGFR < 60 and Pregnancy (N = 1), Low WBC (N = 1), High AST and GGT (N = 2), High ALT and GGT (N = 1), High AST and ALT (N = 1), High AST, ALT and GGT (N = 2), Absence of medium-sized nodule (N = 14), Excluded due to refusal to continue in the study before randomisation and treatment (N = 1), eGFR < 60 and small nodule (N = 1), Pregnant and small nodule (N = 1), High GGT and small nodule (N = 2). ***Met inclusion criteria and randomised but later excluded due to refusal of treatment (N = 1). The participant never received the first dose of treatment.. aFailed to complete treatment due to: High AST and ALT; TA3 (N = 1), High AST and low eGFR; TA1 (N = 1).Reasons for missing participants at follow-ups: 4-months: TA2- travelled (N = 2), TA3- travelled (N = 1), TA4- travelled (N = 1) and dead (N = 1). 6-months: TA1- travelled (N = 4), TA2- travelled (N = 2), TA3- travelled (N = 3), TA4- travelled (N = 4) and dead (N = 1). 18-months: TA1- travelled (N = 3), TA2- travelled (N = 2), TA4- mentally challenged (N = 1) and dead (N = 1). 20-months: TA1- travelled (N = 4), TA2- travelled (N = 3), TA4- travelled (N = 2) and dead (N = 1). N = number; TA = Treatment arm; GGT = gamma-glutamyl transferase; AST = aspartate transaminase; ALT = alanine transaminase; eGFR = estimated glomerular filtration rate.
Baseline characteristics
Participants enrolled and treated had at least one palpable nodule and a baseline skin snip positivity for MF. Baseline characteristics showed no statistical differences between treatment arms for most variables, including sex, body weight, number of IVM rounds, IVM rounds (grouped), nodule counts, nodule site (location), and microfilaridermia intensity (MF/mg skin snip) (Table 1). However, age (p = 0.030) and years lived in the endemic area (p = 0.010) differed between the 4 arms, which occurred randomly during the treatment assignment process. Approximately 12% of participants reported no prior IVM consumption at study onset, but this did not differ between treatment groups or had any effect on treatment outcomes (Table 1).
Adherence to treatment and post-treatment follow-ups
High treatment adherence was observed, with 98.3% (118/120) of enrolled participants completing their assigned treatment regimens per protocol. All participants, including those in the control group/no treatment arm (TA4), were monitored for adverse reactions during the 15-day treatment phase, with a 6-day grace period for missed doses.
Post-treatment follow-ups occurred at 4-, 6-, 18-, 20-month intervals to assess therapeutic efficacy. Out of the 120 participants, retention rates at the four time points were 95.8% (115) at 4 months, 89.1% (106) at 6 months, 95% (113) at 18 months, and 92.4% (110) at 20 months. Skin snip samples were collected at all time points except for the 6-month visit, which focused on IVM administration.
At the 6-month follow-up, IVM was administered to 97.2% (103/106) of the eligible participants. At the 20-month follow-up, 98.2% (108/110) of participants underwent nodulectomy, with two excluded due to absence of palpable nodules. Nodules were successfully excised from 97.2% (105) of participants who consented to the procedure, while no palpable nodules were found during surgery in the remaining 3 participants.
Adverse events following treatment
Adverse events (AEs) were carefully monitored throughout the treatment period to ensure participant safety. RIF and ALB were generally well tolerated, with most AEs classified as mild to moderate. Despite the occurrence of adverse reactions, nearly all participants completed the assigned regimens, with treatment discontinued in two participants due to safety concerns. In addition, seven participants missed one or two doses; however, all completed treatment within the 21-day period. Missed doses were attributable to participant unavailability rather than AE-related complications. Detailed information on treatment interruptions and discontinuation is provided in S2 Table. During treatment, 38 (31.7%) out of the 120 participants recorded AEs (S3 Table). The highest number of participants experiencing AEs was observed in the RIF + ALB – 7 Days group (TA2), where 18 (60%) reported at least one AE. Notably, only one participant (3.3%) in the No Treatment/Control arm (TA4) reported an AE, specifically, elevated AST, despite not receiving any study medication (S3 Fig). A total of 50 AEs were reported, with the highest occurrence in the RIF + ALB – 7 Days group (TA2) at 44.0% (22 AEs), followed by RIF + ALB – 14 Days group (TA1) at 42.0% (21 AEs) (S4 Table).
Overall, 14 different types of AEs were identified. The most frequent AE was reddish-coloured urine (58.0%, 29/50), attributed to RF’s characteristic discoloration. Other common AEs included headache (8.0%, 4/50), fever (4%, 2/50), and elevated levels of ALT (6.0%, 3/50), AST (4%, 2/50) and GGT (4%, 2/50). Less frequent AEs included nausea, diarrhoea, stomach pain, and runny nose. In addition, one participant developed ulceration of the hand and face due to a motor accident, which was classified as a non-treatment related adverse event. Other laboratory abnormalities included low HB (<8 g/dL), low WBC (<3 × 106/µL), and low eGFR (<60 mL/min/1.73m²). Participants were closely monitored until all AEs were resolved.
One serious Adverse Event (SAE) was recorded during the study: a participant in the no treatment arm (TA4) was reported to have committed suicide during the 4-months follow-up. This event was deemed unrelated to the study medications.
Immunohistology
Live versus dead worms
A total of 191 nodules from 89 participants (PP, S5 Table), and 223 nodules from 105 participants (ITT, S6 Table) were examined. In all, 12.0% (23/191) of nodules could not be assessed for viability because they were of non-onchocercal origin (foreign body granulomas, lipomas, lymph nodes). Hence, 168 nodules from 79 participants (PP, S5 Table), and 196 nodules from 92 participants (ITT, S6 Table) were used in subsequent analyses.
The proportion of dead female worms varied across treatment groups, ranging from 25.0% (23/92) in the RIF + ALB – 14 days (TA1) to 36.2% (38/105) in the No Treatment/Control (TA4) group. Statistical analysis using alternating logistic regression (SAS Proc Genmod), which accounts for clustering of worms within patients, revealed no significant differences in the proportion of dead versus live female worms between treatment groups (PP: p = 0.184; ITT: p = 0.219). This analysis accounted for potential dependencies between worms within the same patient, which can arise due to shared host environment and treatment exposure (S5 and S6 Tables).
On average, each nodule contained a total of 2.42 female worms—2.40 living and 2.48 dead female worms for the PP analysis, while the ITT analysis showed an average of 2.30 female worms—2.28 living and 2.38 dead female worms.
For male worms, a lower proportion of mortality was recorded across treatment arms, ranging from 2.8% to 9.7%, lower than the rate reported for female worms. The highest proportion of dead male worms (9.7%, 3/31) was found in the RIF + ALB – 14 days (TA1) group, and the lowest (2.8%, 1/36) in the RIF + ALB – 7 days (TA2) group. As observed for female worms, and accounting for potential dependencies between worms within the same patient, statistical analysis using alternating logistic regression in SAS Proc Genmod revealed no significant differences in the proportion of dead male worms across groups (PP: p = 0.872; ITT: p = 0.838) (S5 and S6 Tables).
On average, each nodule contained a total of 1.15 male worms—1.16 living and 1.00 dead male worms in the PP analysis, while the ITT analysis reported an average of 1.14 male worms—1.15 living and 1.00 dead male worms.
Inhibition of embryogenesis
The study evaluated the effect of the treatments on O. volvulus embryogenesis by comparing the proportions of adult female worms with normal and degenerated embryos across treatment groups. The impact of the treatment regimens on embryogenesis was evaluated using an alternating logistic regression model (S8 and S10 Tables). The proportions of living female worms with normal embryogenesis were 28.6% (18/64), 32.9% (24/79), 32.7% (16/58) and 30.5% (18/63) in the RIF + ALB – 14 Days (TA1), RIF + ALB – 7 Days (TA2), ALB alone – 14 Days (TA3) and No Treatment/Control (TA4) groups (S7 Table).
Alternating logistic regression analysis showed no statistical difference in embryogenesis between the treated groups (TA1, TA2, TA3) and the control group (TA4) (p > 0.05) (S8 Table). However, the RIF + ALB – 7 Days (TA2) and the ALB alone – 14 Days (TA3) groups had a 7.9% and 7.2% reduction in the proportion of female worms with normal embryogenesis, compared to the No Treatment/Control group, respectively. Similarly, the RIF + ALB – 14 Days (TA1) group showed a 6.2% reduction in the proportion of female worms with normal embryogenesis compared to the No Treatment/Control group.
In addition to embryogenesis, another indicator of female worm fecundity—the presence of free MF in nodule tissues—was also evaluated. Like the findings on embryogenesis, no differences were observed between the four treatment groups (p = 0.571, S11 Table).
Both PP and ITT analyses showed no statistically significant differences between the treatment groups and the control group, confirming the robustness of the primary analysis and minimising potential bias due to non-compliance with the treatment regimen or loss to follow-up after treatment initiation (S9–S10 Tables).
Effect of study medications on Wolbachia depletion
The Wolbachia loads in living O. volvulus were assessed semi-quantitatively and classified as ‘many’, few’ and ‘none’ as previously described [47]. In total, 168 nodules from 79 individuals were analysed in the PP group (S14 Table). In the ITT group, 196 nodules from 92 study participants were analysed (S15 Table). The proportion of living female worms containing Wolbachia was not reduced in any of the treatment arms compared to the No Treatment/Control arm (S14 and S15 Tables). Even though 4.8% of living female worms in the RIF + ALB – 14 Days (TA1) had no Wolbachia (S14 Table), statistical analysis revealed no significant reduction in Wolbachia load in this group compared to the control group, where 1.6% of female worms had no Wolbachia.
For male worms, there was no statistical difference in Wolbachia reduction in the treated arms when compared to the No Treatment/Control arm for male worms that could be assessed for Wolbachia (S16 and S17 Tables). The Wolbachia assessment methods were the same for both male and female worms.
Real-time quantitative PCR (qPCR)
Wolbachia depletion in skin microfilariae
Traditionally, anti-Wolbachia drug efficacy evaluations have relied on immunohistological examination of nodulectomised samples, a method limited by its invasiveness and single time point assessment throughout a study. Real-time quantitative PCR (qPCR) targeting Wolbachia DNA in skin MF offers a less invasive surrogate, enabling longitudinal monitoring of Wolbachia depletion and early identification of sub-optimal drug responses in a study.
In this study, qPCR assays amplified the single-copy Wolbachia ftsZ gene (wOvFtsZ) and the O. volvulus actin gene (OvActin). The ratios wOvFtsZ/MF and wOvFtsZ/OvActin were calculated to assess treatment efficacy at 4-months post-treatment. Samples were included if microscopy confirmed at least 1 MF count and OvActin was detected by qPCR. Four MF-positive samples (microscopy) that were OvActin negative across the treatment arms except the no treatment/control arm (TA4) were excluded from the Wolbachia depletion analysis (Table 2).
The qPCR results indicated no statistical differences in OvActin copy numbers across treatment arms (p > 0.05) when MF DNA was amplified from skin snips obtained 4-month post-treatment.
Changes in Wolbachia levels post-treatment were calculated as percentage median reductions relative to the no treatment control. No differences in wOvFtsZ/MF ratios were observed between the treated and the control arms (Figs 2A and S4A). However, a reduction in this index was observed between the RIF + ALB – 7 Days (TA2) and RIF + ALB – 14 Days (TA1) (PP: p = 0.015), with TA1 showing a 17.79% reduction.
TA = Treatment arm; PP = per-protocol; TA1 = RIF + ALB – 14 days; TA2 = RIF + ALB – 7 days; TA3 = ALB alone – 14 days; TA4 = No treatment/control. Graphs show the median values and interquartile ranges. Kruskal-Wallis with Dunn’s multiple comparison tests, with Holm-Bonferroni correction applied, calculated using Stata version 16. The following p-values indicate statistically significant differences before correction, but did not remain significant after correction: wOvFtsZ/MF: TA2 vs TA3, p = 0.026, TA1 vs TA4, p = 0.029; wOvFtsZ/OvActin: TA2 vs TA1, p = 0.014.
The wOvFtsZ/OvActin ratio revealed a more pronounced treatment effect. A median reduction in Wolbachia load was observed in the RIF + ALB – 14 Days (TA1) at a rate of 47.91%, compared to the No Treatment/Control group (TA4) (PP: p = 0.018). All other treatments were less effective than the 14 Days RIF + ALB regimen in reducing Wolbachia loads (p > 0.05) (Figs 2B and S4B). Additionally, RIF + ALB – 14 Days (TA1) also had lower wOvFtsZ/OvActin Wolbachia loads compared to ALB alone – 14 Days (TA3) (PP: p = 0.022), indicating superior efficacy of the 14 Days RIF + ALB combination over ALB alone – 14 Days monotherapy (Figs 2A and S4A), potentially indicating the action of RF.
Wolbachia depletion from nodules sections
Real-time qPCR was used to support immunohistological findings by quantitatively assessing Wolbachia depletion in worms in nodule tissue sections (S18 Table). O. volvulus DNA was extracted from 8 consecutive sections per nodule, adjacent to sections used for histology to maximise DNA yield and analysed for Wolbachia FtsZ and O. volvulus actin. Out of the 196 nodules evaluated by immunohistology, DNA was successfully isolated from 152 nodules. The remaining nodules were calcified and had been treated with EDTA for immunohistology, making DNA extraction from them not possible. Due to variations in the amount of human tissue relative to worms, and between nodules, Wolbachia loads were normalised to O. volvulus actin levels following the method described by Gilbert et al., (2005) [51]. Nodules containing only dead worms, confirmed by histology or lacking detectable actin signals were excluded from further analysis (PP: n = 20; ITT: n = 22). The absence of an actin signal likely indicates absence of worms in the nodule section.
The qPCR results (S18–S20 Tables) generally corroborated the immunohistology findings, showing no differences in Wolbachia depletion in most treatment arms, compared to the control. Normalised median Wolbachia loads (wOvFtsZ/OvActin) increased by 42.69% in RIF + ALB – 14 Days (TA1) and 28.65% in RIF + ALB – 7 Days (TA2), while ALB alone – 14 Days (TA3) showed a reduction of 21.05%. However, these changes were not significant (p > 0.05).
Changes in microfilarial densities following treatment
Microfilarial density, measured as MF/mg skin snip, serves as the standard measure of infection intensity. At baseline, no differences in the microfilarial loads (MF/mg skin snip) were observed across all treatment arms (p = 0.678). At 20-months post-treatment, the highest microfilarial density was observed in the No Treatment/Control group (TA4), while the lowest was recorded in ALB alone –14 days (TA3). Despite the observed variations, there were no statistical differences in microfilaridermia between all arms at 4-, 18- and 20-months post-treatment assessment (Table 3).
Longitudinally, a slight increase in skin MF/mg skin snip was observed 4 months post-treatment compared to baseline in the ALB alone– 14 days group (TA3) (p = 0.018) (Table 3). However, reductions were observed at 18- (p = 0.007) and 20-months (p = 0.003), indicating a delayed but substantial decline in microfilarial densities following treatment (Table 3). Similarly, an increase in MF/mg skin snip was detected in the No Treatment/Control group (TA4) at 4 months in the PP analysis (p = 0.034; Table 3). However, this increase was not significant in the ITT analysis (S22 Table) and was not sustained at the 18- and 20-month time points, indicating that the rise at 4-months was transient.
Discussion
This study represents the first clinical evaluation of high-dose Rifampicin (RIF) combined with Albendazole (ALB) as an alternative regimen for onchocerciasis in humans. This is in response to the failure of IVM in clearing MF in areas with decades of the treatment, potentially due to IVM resistance or sub-optimal parasite clearance [52,53]. Potential adulticidal drugs such as high-dose RIF plus ALB targets adult worm embryogenesis, leading to reduction or clearance of skin MF, aiming to complement efforts to achieving the WHO 2030 elimination target [4].
In this current trial, the safety profile of high-dose RIF in combination with ALB, was favourable. Adverse events were generally mild and included expected effects such as reddish urine discoloration, headache, and transient fever. Temporary elevations in liver enzymes were also observed but resolved spontaneously, consistent with previous pharmacokinetic and pharmacodynamic studies on the safety of short-course, high-dose RIF [12,34]. Concerns regarding resistance development, particularly given RIF’s role in tuberculosis treatment, are mitigated by the short duration of administration for filarial indications and the absence of evidence linking short-term use to clinically significant resistance [12]. It should be noted that no pharmacokinetic bioanalysis was performed; therefore, steady-state drug exposure and potential drug–drug interactions could not be confirmed and remain speculative in the context of this study.
In evaluating the macrofilaricidal efficacy of the combination therapy (high-dose RIF plus ALB), we did not observe a statistically significant increase in adult worm mortality or inhibition of embryogenesis, compared to the control arm. These outcomes contrast with preclinical studies that demonstrated significant Wolbachia depletion and partial reductions in female worm burden with similar regimens [12,25], and are likewise inconsistent with the findings of our lymphatic filariasis (LF) trial, using the same regimen [54]. In our LF clinical trial, we observed an early, sustained and significant reductions in circulating filarial antigen (CFA) levels in the RIF plus ALB groups even though there was no consistent and sustained decline in CFA negativity across time points beyond 4 months [54].
The limited efficacy observed in the current study may be attributed to several factors. RIF, despite achieving high tissue concentrations, has a short half-life (2–5 hours) which may necessitate frequent dosing to maintain therapeutic levels [55], partly informing the rationale for administering the regimen for a minimum of 7 days. ALB absorption is variable and influenced by dietary fat intake [56]; therefore, participants were instructed to eat prior to their daily treatment dose. It is acknowledged that food intake enhances ALB absorption [57,58], and RIF is generally recommended to be taken on an empty stomach [32,59]. However, co-administration of RIF and ALB was done in this study to reduce the tendency of stomach upsets or nausea; a choice that reflects the real-world administration conditions anticipated for its potential use as an MDA drug [60,61]. Although pharmacokinetic assessments were not conducted, we considered that variability related to food intake would have minimal impact on drug bioavailability, as treatments were administered within participants’ communities around mid‑morning. Nevertheless, because the interval between food consumption and treatment dosing was not recorded, RIF bioavailability may have been affected in participants who ate within 2 hours prior to drug administration.
It should be noted that the same treatment modalities were applied in our LF trial [54], which demonstrated marked efficacy of the drug regimen against W. bancrofti infection. The contrasting outcomes between these two trials more likely reflect biological differences between O. volvulus and W. bancrofti. Nodular tissue compartmentalisation, greater worm biomass, and potentially differing Wolbachia susceptibility thresholds in O. volvulus may serve as barriers to achieving comparable macrofilaricidal effects. Importantly, Turner et al. (2017) reported that RIF did not adversely affect ALB bioavailability under controlled conditions; however, their study was conducted in mice [25]. Human-specific metabolic pathways may differ from those in preclinical models, and interindividual variation in drug metabolism, including variation across different ethnic backgrounds, may further influence systemic drug exposure in clinical settings [62,63]. Since no pharmacokinetic assessment was conducted in this trial, the systemic exposure levels of both drugs remain unknown and can only be assumed to have been near optimal.
Systemic drug distribution and nodule vascularisation in humans may also differ from preclinical models, potentially limiting drug penetration and reducing effective concentrations at target sites. The ‘nodular sanctuary effect’, in which dense fibrous tissue in onchocerciasis nodules limits drug access, is a recognised barrier to achieving significant macrofilaricidal activity [64–66], and may have contributed to insufficient intranodular drug concentrations, thereby failing to disrupt female worm reproductive functions effectively. It is also important to acknowledge that the preclinical model used to define the RIF regimen evaluated in this trial may not fully represent the biological and pharmacological complexity of adult O. volvulus infections in humans. The male O. ochengi implant model in SCID mice [12] differs from the human clinical setting in parasite species, worm biomass, parasite location, and nodular tissue architecture. Indeed, no reliable small animal model of microfilarial O. volvulus infection currently exists [67], which limits the accuracy of pharmacokinetic-pharmacodynamics predictions derived from this surrogate model [2]. This translational gap highlights the need for empirical dose-ranging studies in humans that incorporate pharmacokinetic assessment to establish the drug exposure levels required for effective anti-Wolbachia activity in the biological context of human onchocerciasis. Studies utilising longer treatment durations (4–6 weeks) with alternative anti-Wolbachia agents, such as DOX alone or in combination with ALB, have demonstrated superior effects on adult worm viability and embryogenesis [16,68].
Male worms exhibited consistently lower mortality rates across all treatment arms, likely due to their reduced dependence on Wolbachia for survival, a finding consistent with prior studies [25,36]. Female worms, which rely heavily on Wolbachia for reproduction and survival, showed higher mortality rates, though the observed effects were modest, compared to preclinical outcomes.
Regarding RIF’s potential to deplete Wolbachia in O. volvulus, our findings present a complex picture. At 4 months post-treatment, the 14-day RIF + ALB regimen achieved a statistically significant 47.9% reduction in Wolbachia load in skin MF as determined by qPCR. Although this was lower than the > 90% reductions reported in preclinical studies using high-dose RIF in adult worms of mouse models [12,25], Wolbachia depletion in MF nevertheless provides a useful surrogate for adult worm histology, and indicates measurable anti-Wolbachia activity in humans. It is noteworthy, however that skin MF are dermally resident and therefore directly accessible to systemically circulating drug levels, without the physical barrier of fibrous nodular tissue. The sub-optimal Wolbachia depletion in skin MF observed at 4 months may be suggestive of lower than expected systemic RIF exposure levels required for achieving >90% Wolbachia depletion, as defined by Aljayyoussi et al. (2017) [12]. However, this observation is inconsistent with earlier DOX studies in onchocerciasis and LF that showed >90% Wolbachia depletion necessary for a sustained anti-Wolbachia effect [11,47,69]. By 20 months post-treatment, immunohistological evaluation of extirpated nodules revealed no significant Wolbachia depletion in adult worms across treatment arms relative to the No Treatment/Control arm. This finding is consistent with recent experimental evidence showing that Wolbachia can rebound to baseline levels within months of antibiotic treatment in preclinical models [70]. One proposed mechanism for this recrudescence involves Wolbachia populations sheltered within ovarian sheath cells that may be less accessible to antibiotic penetration [71]. While this remains a plausible explanation, the hypothesis is still preliminary, as prior anti-Wolbachia regimens using DOX achieved >90% depletion [16], an efficacy anticipated from RIF plus ALB combination therapy. The contrasting outcomes between skin MF and adult worms likely reflect differences in parasite stage, tissue compartmentalisation, and drug pharmacokinetics. While RIF and ALB may reach skin microfilariae effectively, drug penetration into fibrous nodules housing adult worms remains a key unresolved challenge [65,66]. Moreover, the 14-day treatment duration may have been insufficient to sustain therapeutic drug levels long enough for durable Wolbachia depletion, particularly within nodules [72]. Collectively these findings underscore the need to reassess dosing strategies and to explore formulations or delivery systems that enhance drug retention and tissue penetration.
To improve the therapeutic potential of RIF + ALB, adjustments to dosing regimens may be required. Extending RIF treatment beyond 14 days, and as suggested by Specht et al. (2008), combinatory therapy other than ALB such as oxfendazole which has demonstrated good synergistic effects with other drugs could allow for a more sustained Wolbachia depletion and increase the likelihood of worm death [35]. Likewise, other formulations with enhanced bioavailability and tissue penetration such as benzimidazole may strengthen the synergistic anti-Wolbachia effect when combined with RIF [73].
Regarding the effects of the study medications on skin microfilarial loads, baseline microfilarial densities were comparable across all arms. It is important to note that neither RIF nor ALB exerts direct microfilaricidal activity. RIF acts indirectly by depleting Wolbachia endosymbionts within adult female worms, which subsequently disrupts embryogenesis and leads to gradual decline in MF production over time [12,25]. ALB and other benzimidazole similarly act indirectly, primarily by inhibiting β-tubulin polymerisation in nematodes, which disrupts microtubule formation and interferes with the female embryogenesis pathway, rather than directly killing existing MF in the skin [22,74]. Meaningful reductions in skin microfilarial loads from either drugs are therefore dependent on the extent and durability of their effects on adult worm reproductive function [16]. In the ALB alone (14-day) group, significant reductions were observed at 18- and 20-months post-treatment, though not at 4-months, where a slight but significant increase was seen, suggesting a delayed but substantial response to ALB treatment over time. The combination regimens did not achieve reductions comparable to those reported for ivermectin, which has been shown to deplete over 99% of MF within 1–2 months after administration [75,76], and differences between the RIF plus ALB arms and the No Treatment arm differences were not statistically significant. The limited efficacy of the combination regimens is consistent with Richards et al., (2007), who found that RIF-based regimens did not significantly reduce microfilarial loads at 9 months post-treatment [36], reinforcing the broader principle that antibiotics alone do not rapidly clear MF because they act indirectly through Wolbachia depletion rather than through direct microfilaricidal activity [16]. Meaningful early reductions in MF are therefore more likely when antibiotics are combined with a direct microfilaricidal agent such as IVM, as demonstrated in previous DOX plus IVM combination studies [16,77].
The reductions in microfilarial densities observed in the ALB alone (14-day) treatment arm reaffirm its potential as a standalone therapy in specific contexts [37]. Improved drug bioavailability and host immune responses, in the absence of pharmacokinetic interference from RIF, likely contributed to this outcome. RIF is a potent inducer of cytochrome P450 enzymes and may accelerates ALB biotransformation, thereby reducing systemic ALB exposure and potentially diminishing efficacy during co-administration [74,78], an interaction that may have undermined the combined effects of RIF + ALB in this trial.
The increase in microfilarial densities observed in TA4 (No treatment) at 4-months is consistent with the natural dynamics of O. volvulus, where adult worms continuously produce MF in the absence of intervention. The observed fluctuations in microfilarial counts in this arm across timepoints likely reflect the interplay of ongoing parasite reproduction, seasonal transmission dynamics [79], and host immune modulation [80], rather than any treatment effect. While similar patterns of stable or increasing microfilarial loads in untreated individuals have been reported in IVM-naïve populations in Cameroon [53,81,82], it is worth noting that the IVM dose administered at the 6-months follow-up visit may also have contributed to the variability observed in microfilarial counts at subsequent timepoints in the No treatment arm. It is also notable that the significant increase in the no-treatment arm was observed only in the PP analysis and not in the ITT analysis, suggesting this finding should be interpreted with caution.
Anti-Wolbachia therapy has been validated as a macrofilaricidal approach across filarial infections, but important differences exist between O. volvulus and the lymphatic filariae. In LF, the same high-dose RIF plus ALB regimen produced evidence of macrofilaricidal activity against W. bancrofti in our Ghana trial [54]. By contrast, earlier field studies in onchocerciasis found no significant macro- or microfilaricidal effects from short-course RIF regimens [36], and even 4-week RIF treatment only modestly outperformed shorter courses [35]. DOX has shown more consistent macrofilaricidal and sterilising effects across multiple filarial systems, including O. volvulus [16,68], W. bancrofti [83,84], and B. malayi [85], though requiring 4–6 weeks of treatment. Taken together, these observations indicate that while LF species such as W. bancrofti and Brugia respond robustly to short-course anti-Wolbachia regimens, O. volvulus appear more refractory, requiring longer or more intensive antibiotic exposure to achieve sustained Wolbachia depletion. This highlights the need for new short-course macrofilaricidal candidates.
A key limitation of our study was the inability to assess Wolbachia depletion in skin MFs at 18-and 20-months post-treatment, primarily due to resource constraints. Although sustained Wolbachia depletion was not observed in adult worms at 20 months, corresponding longitudinal data from MFs would have enabled direct comparison across parasite life stages, assessment of potential bacterial recrudescence, and deeper insights into the temporal dynamics of Wolbachia depletion. The absence of these measurements therefore limited a more comprehensive evaluation of long-term treatment effects. As discussed earlier, the lack of pharmacokinetic assessment means it cannot be confirmed whether the target steady-state RIF exposure was achieved in trial participants, and the absence of RIF monotherapy arms precludes evaluation of whether drug-drug interactions between RIF and ALB may have adversely influenced treatment outcomes. Together, these represent significant limitations that hamper definitive conclusions about the intrinsic efficacy of the RIF plus ALB regimen for onchocerciasis. Nonetheless, the study provides important insights into the short-term effects of high-dose RIF combined with ALB and highlights the need for strategies capable of achieving more durable therapeutic efficacy.
In conclusion, this study confirms the safety of high-dose RIF in combination with ALB for treating onchocerciasis. The limited macrofilaricidal effects observed suggest that treatment duration and pharmacokinetic properties were insufficient to achieve sustained Wolbachia depletion, and subsequent adult worm death or inhibition of embryogenesis. These findings highlight the need for longer or optimised combination regimens, as well as incorporation of pharmacokinetic assessments in future trials to verify target drug exposure levels. The reductions in microfilarial densities observed in the ALB alone (14 days) treatment arm reaffirm its potential as a standalone therapy in specific contexts. Translating the preclinical success of RIF plus ALB combination therapy into clinical efficacy for onchocerciasis remains a challenge, and future studies should evaluate whether extended RIF courses, alternative combination regimens, or as improved benzimidazole formulations with enhanced bioavailability and tissue penetration, can strengthen anti-Wolbachia effects and complement efforts towards onchocerciasis elimination.
Supporting information
S1 Fig. qPCR DNA quantification standard curve for gBlock (OvActin) at a 10-fold dilution.
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S2 Fig. Participant flow chart showing treatment allocation, presence/absence during follow-up visits, data analysis set.
ITT and/or PP analysis sets, and reason for absence or exclusion from PP analysis.
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S3 Fig. Adverse Events experienced by participants.
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S4 Fig. Effects of study drugs against Wolbachia in skin microfilariae at 4 months post-treatment—ITT analysis.
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S5 Fig. Changes in microfilarial densities 4-, 18-, and 20- months following treatment—PP analysis.
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S6 Fig. Changes in microfilarial densities 4-, 18-, and 20- months following treatment—ITT analysis.
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S1 Table. Primers, Probes and Standards (gBlocks) sequences used for the real-time qPCR.
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S2 Table. Participant compliance with treatment regimens.
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S4 Table. Adverse event assessment, detailed.
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S5 Table. Effect of treatment on adult worm—PP analysis.
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S6 Table. Effect of treatment on adult worms—ITT analysis.
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S7 Table. Effect of treatment on embryogenesis—PP analysis.
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S8 Table. Effect of treatment on embryogenesis: statisticsa,b—PP analysis.
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S9 Table. Effect of treatment on embryogenesis—ITT analysis.
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S10 Table. Effect of treatment on embryogenesis: statisticsa,b–ITT analysis.
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S11 Table. Effect of treatment on intranodular microfilariae—PP analysis.
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S12 Table. Effect of treatment on intranodular microfilariae (MF)—ITT analysis.
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S13 Table. Effect of treatment on the presence of Wolbachia in female worms: immunohistology—PP analysis.
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S14 Table. Effect of treatment on the presence of Wolbachia in female worms: immunohistology—ITT analysis.
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S15 Table. Effect of treatment on the presence of Wolbachia in male worms: immunohistology—PP analysis.
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S16 Table. Effect of treatment on the presence of Wolbachia in male worms: immunohistology—ITT analysis.
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S17 Table. Selection of skin snip samples for qPCR and Wolbachia depletion analysis—ITT analysis.
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S18 Table. Effect of treatment on the presence of Wolbachia in nodule sections: qPCRa—PP analysis.
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S19 Table. Effect of treatment on the presence of Wolbachia in nodule sections: statisticsa,b—PP analysis.
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S20 Table. Effect of treatment on the presence of Wolbachia in nodule sections: PCRa—ITT analysis.
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S21 Table. Effect of treatment on the presence of Wolbachia in nodule sections: statisticsa,b—ITT analysis.
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S22 Table. Changes in microfilarial densities 4-, 18-, and 20- months following treatment —ITT analysis.
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S23 Table. Iron deposition in adult worm—PP analysis.
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S24 Table. Iron deposition in adult worm—ITT analysis.
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S25 Table. Viability status of adult worm —PP analysis.
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S26 Table. Viability status of adult worm—ITT analysis.
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Acknowledgments
We thank the community leaders and members of the study communities in the study districts for their cooperation and participation. We also acknowledge the support of community health volunteers, community nurses, and other health personnel who assisted the study in various ways. We are grateful to the District, Regional, and Ghana Health Service for their support throughout the study.
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