Abstract

Author summary

Citation: Kumar P, Choi Y-J, Fischer K, Hedtke SM, Kode A, Opoku N, et al. (2025) Genetic structuring and estimation of reproductive adults in Onchocerca volvulus: A genome-wide analysis across hosts and regions. PLoS Negl Trop Dis 19(7): e0013221. https://doi.org/10.1371/journal.pntd.0013221

Editor: James Lee Crainey, Instituto Leonidas e Maria Deane / Fundacao Oswaldo Cruz, BRAZIL

Received: December 13, 2024; Accepted: June 9, 2025; Published: July 1, 2025

Copyright: © 2025 Kumar 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 NCBI SRA repository under BioProject PRJNA1189144. Individual sample information is provided in S2 Table.

Funding: This work was supported by the US National Institutes of Health [https://www.nih.gov/; grant number AI144161 to MM and WNG] and the Bill and Melinda Gates Foundation [https://www.gatesfoundation.org; grant number GH5342 to PUF, grant number OPP1190749 to PUF]. The funders had no role in the study design, data collection, analysis, interpretation, or manuscript writing.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Onchocerciasis, or river blindness, is caused by the filarial nematode parasite Onchocerca volvulus. Infection may cause pathologies including skin de- or hyper-pigmentation, severe dermatitis, pruritus, epilepsy, Nakalanga syndrome, irreversible blindness, and social ostracization [1,2]. It is a neglected tropical disease (NTD) and the second-leading cause of irreversible blindness worldwide. In 2022, the World Health Organization (WHO) estimated that at least 246 million people lived in communities that should be targeted for mass drug administration with ivermectin (MDAi) [3]. Ivermectin kills most of the first stage larvae (microfilariae, mf) in the skin and is embryostatic, acting as a temporary contraceptive for female worms. MDAi thus interrupts the transmission cycle because there are no mf for the blackfly vectors to transmit [4–6], and is therefore the main strategy used in efforts to eliminate onchocerciasis [7,8]. To date, four countries in the Americas (Colombia, Ecuador, Guatemala, and Mexico) have been WHO-verified for having successfully eliminated transmission of onchocerciasis [3,9]. In sub-Saharan Africa, where >99% of people who are at risk of onchocerciasis reside, over 160 million people were treated in 2022, 16 countries achieved 100% geographical coverage, and Niger has submitted a dossier for verification of elimination [3].

Rapid Epidemiological Mapping of Onchocerciasis (REMO) was initially used to identify meso- and hyperendemic O. volvulus foci that were prioritized for intervention [10]. Adult female O. volvulus form subcutaneous nodules, known as onchocercomas, that can be readily palpated. Nodules that are not externally visible or palpable are sometimes located in deeper tissues and are often attached to bones [11–13]. Male worms migrate between nodules, where reproductively active, sessile females reside [14]. Fertilized female worms release thousands of mf into the skin [11], which then disperse throughout the body (although the rate of dispersal is not known). Skin snips are often taken from four different parts of the body (e.g., the iliac crest and calf) to assess the presence of skin mf. The number of mf in a skin snip can be counted as a proxy for worm burden; however, such counts do not account for variability in adult reproductive output or in immune response to mf, and may not accurately reflect the true adult worm burden.

In Africa, there remain areas where transmission persists despite MDAi for longer than the ~ 15-year lifespan of the adult worms, or where transmission has resumed after MDAi has been stopped [6,15–21]. Furthermore, so far little or no MDAi has been performed in areas with hypoendemic onchocerciasis or in areas coendemic for loiasis where there is an elevated risk of serious adverse events after administration of ivermectin. Ongoing transmission can be associated with the operational challenges involved in running decades-long MDAi programs, particularly in areas with high onchocerciasis endemicity requiring high coverage and many repeated rounds of ivermectin. In addition, sub-optimal responses of adult female worms to the contraceptive effect of ivermectin and sub-optimal mf killing of ivermectin have been reported [22–31]. Transmission can also be re-introduced to a focus after MDAi has been stopped due to either migration of infective vectors of O. volvulus (blackflies in the genus Simulium) from a neighboring focus in which transmission continues, or when infected people move between communities (often for farming work) [16,32,33].

Genetic epidemiology can be applied to investigate ongoing transmission despite MDAi and post-MDAi recrudescence [34]. Through longitudinal sampling of mf, genomic approaches can distinguish between adult worms that continue to reproduce after multiple rounds of therapy and new infections resulting from ongoing transmission, while also tracking the decline in female reproduction over time—a critical challenge in evaluating the impact of MDAi [35]. This strategy would enable direct monitoring of drug efficacy on female fertility during clinical trials and MDAi, a capability that is currently unavailable. Population genetic data has been used to explore how parasites have moved throughout the landscape in West Africa [24,36,37]. In Ghana, for example, similarities in genetic variation between river basins in a central transect spanning the ecological transition zone between savannah and forest ecosystems suggested that parasites have been moving via infected people or infective vectors throughout a 250-km transect [24,37,38]. However, worms sampled from Ghana are genetically distinct from worms from Mali, Cote d’Ivoire, Cameroon, and the Democratic Republic of the Congo (DRC) [24,37,39], suggesting that people and vectors are not regularly moving between Ghana and other countries. Population genetic data has also been used to estimate the number of reproductively active female worms in a person and has shown that the worm burden is higher in South Sudan than in the DRC [39]. Tracking the number of reproductively active female worms may be more informative about the success of ongoing MDAi than counts of the mf burden in the skin [39].

Previous population genetic research, however, has focused on either very small sample sizes within a country [36], pooled genome coverage with low depth per worm [24], or exclusively on mitochondrial data [37,39]. Mitochondrial data are limited, as they can only track maternal lineages (mitochondria are maternally inherited in O. volvulus), are inherited as a single locus, and have far fewer variable sites than the much larger nuclear genome inherited from both parents. Here, we demonstrate the increased resolution possible using nuclear genome data from mf to evaluate genetic variation and population structure in O. volvulus. The goals of this study were to i) investigate the nuclear and mitochondrial genetic differentiation among mf within hosts, between hosts within a country, and between countries, ii) estimate the minimum number of reproductively active adult female and male worms in a host based on mf genotypes and iii) examine the genetic diversity and patterns of LD in the autosomes and X-chromosome in O. volvulus using high-coverage diploid genome data. These analyses were facilitated by whole-genome sequencing of mf sampled from participants in historical samples or clinical trials in West Africa (Liberia and Ghana) and clinical trials in Central Africa (eastern DRC).

Results

Discussion

In this study, we achieved high nuclear genome coverage through whole-genome amplification of O. volvulus mf, the larval stage routinely collected for diagnosis. Obtaining sufficient quantity and quality of DNA from single mf samples for comprehensive nuclear genome analysis has long been a challenge. Using our genome-wide nuclear genetic data from samples from Ghana, Liberia, and DRC, we have (i) examined the patterns of genetic structuring within and between hosts as well as between countries, (ii) estimated the number of reproductively active adult female and male worms in individual hosts, and (iii) assessed the genome-wide patterns of genetic variation across the different chromosomes in O. volvulus. Historically, the small physical size of mf (250–360 μm ×ばつ 5–9 μm) [45], with an estimated <250 nuclei, and the variable (and often suboptimal) quality of sample preservation have limited the generation of high-quality sequencing data and full genomic analysis in O. volvulus mf. Using whole genome amplification with improved sample preservation, processing, and library construction for filarial nematodes that we have developed, validated and recently reported [35,46], we generated a WGS dataset (315 individual mf; 175, 37, and 103 samples from Ghana, Liberia, and DRC, respectively) with significantly improved nuclear genome coverage, achieving a median breadth of coverage of 94%. However, the quality of sequencing data varied between samples collected in different clinical trials conducted in different countries. Overall, nuclear genome coverage was higher for West African samples compared to DRC samples, likely due to variation in sample preservation. West African samples were stored in RNAlater at -20°C and shipped cold, while DRC samples were stored in ethanol and shipped at ambient temperature.

Analysis of genetic variation among mf samples can provide insights into population connectivity and historical gene flow useful for delineating transmission zones [34]. To date, mitochondrial genetic variation has been the primary genetic marker used in these population studies in O. volvulus [37–39]. Our dataset enabled, for the first time, a systematic comparison of patterns between nuclear and mitochondrial genetic variation in O. volvulus mf. Using autosomal SNPs that represent a large number of independent loci, it was possible to genetically differentiate parasite populations between hosts and between geographically close countries with higher resolution than mitochondrial haplotype-based analysis. Assigning individual worms to their source populations was more successful at both the individual host and country levels using allele-sharing patterns in the nuclear genome. These results indicate that, compared to mitochondrial data, nuclear genomic data (when combined with population sampling appropriate for the study aim) provide a higher resolution analysis that can capture more fine-grained genetic structure in the parasite population. Detecting such differences is essential for analyses aiming to detect shorter-distance parasite migration via host/vector movement. Compared to the mitochondrial genome, which is maternally inherited as a single locus (linkage group) without recombination, analysis of the sexually recombining nuclear genome can reveal admixture events and quantify the populations’ ancestry compositions. In addition, genome-wide analysis of nuclear genetic variation could provide deeper insight into the impact of MDA on parasite populations (e.g., selection of genotypes that provide survival/reproductive advantage in response to treatment), contributing to more effective and sustainable intervention strategies.

Genetic data from mf can be used to obtain information that is epidemiologically important but difficult to obtain through other methods, such as estimates of adult worm burden and fecundity in individual hosts [35,39]. The success of transmission interruption through MDA depends entirely on the cumulative decline in female fecundity over time due to repeated ivermectin treatments, but there is currently no direct method to measure this decline. While treatment does not reduce the number of palpable nodules, the presence of skin mf indicates that at least one fertile female remains, without providing a clear measure of progress toward sterility—information critical for assessing MDA progress and making decisions about when to stop treatment. Monitoring changes in reproductively active adult worm populations through longitudinal sampling of mf would help assess the effects of treatment, identify new infections, and detect treatment failures.

The first study using genetics to estimate the number of reproductive adults in O. volvulus by sequencing microfilariae was limited by using only mitochondrial data, which only represents reproductive females [39]. In this study, we estimated the number of both male and female reproductively active adults using nuclear genetic data and evaluated whether this approach improves estimates of worm burden. Our method for estimating adult worm counts from mf data was based on a sibship reconstruction approach previously used for Wuchereria bancrofti and Brugia malayi, the filarial species that cause lymphatic filariasis [35]. Sibling relationships were estimated using autosomal SNPs, and maternal or paternal sibling groups were assigned based on the segregation patterns of mitochondrial haplotypes, X-linked (excluding PAR), and Y-linked variants. We assessed whether microfilariae sampling was sufficient to detect the number of contributing parents using rarefaction and extrapolation analyses (S7 and S8 Figs) [35,39]. The estimated worm burden, and consequently the optimal number of microfilariae to sequence, varied between individuals and across countries (Figs 3 and S7), likely due to differences in age, exposure to infected blackflies based on time spent outdoors, and community-level transmission intensity. The number of maternal sibling families inferred from nuclear data, representing the number of reproductively active adult females, was lower than the number of unique mitochondrial haplotypes in 5 out of 13 participants. Given that mitochondrial DNA is maternally inherited in O. volvulus, we expected all maternal siblings within a host to share identical mitochondrial haplotypes. However, in 5 participants, mf with different mitochondrial haplotypes exhibited close autosomal relatedness, suggesting full or half-sibling relationships. Upon analyzing the genetic distance between these mitochondrial haplotypes, we found only a single nucleotide substitution, indicating possible genotyping errors. Consequently, these mitochondrial haplotypes were grouped into a single maternal family. To reduce the risk of erroneously merging distinct maternal sibling families (e.g., paternal half-siblings), we further examined the inheritance patterns of X-linked haplotypes among male mf to ensure consistency with the expectation that no more than two maternally inherited haplotypes segregate among maternal siblings. This cross-analysis of mitochondrial and X-linked haplotypes could also help identify cases where multiple adult females share the same mitochondrial haplotype, such as a high-frequency ancestral haplotype within the population.

Because whole-genome sequencing after DNA amplification using phi29 polymerase can increase genotyping errors [47], we applied stringent filtering to remove false-positive variants, including the exclusion of singleton SNPs prior to mitochondrial haplotype construction (see Methods for details). While determining the true genotyping error rate in amplified WGS data is challenging, these errors are likely to disproportionately affect nuclear and mitochondrial-based parentage inference. For sibship inference, a large number of independent SNPs were used to estimate genome-wide relatedness, making this analysis less vulnerable to genotyping errors introduced by genome amplification [35]. In contrast, the accuracy of mitochondrial haplotype determination is likely more sensitive to genotyping accuracy, as even a single substitution in the genome can result in distinct haplotypes. In addition to genotyping uncertainty, there are other possible explanations for the discordant patterns between mitochondrial and autosomal relatedness, such as inaccurate inference of sibling groups from autosomal data and unequal segregation of heteroplasmic mitochondrial genomes from an adult female to her progeny. Furthermore, identifying sibship groups through autosomal DNA comparison can be imprecise, as genetic relatedness is a continuous measure that may not perfectly align with theoretical expectations based on pedigree relationships. These variations depend on factors such as the number of chromosomes and their crossover rates [48].

An experimental approach to investigate the causes of these discrepancies would involve genotyping adult worms (somatic tissue) and in utero mf from nodules. Comparing the mitochondrial haplotypes of individual adult females to their intrauterine mf would be especially informative in determining whether multiple mitochondrial haplotypes can occur among the mf from a single female due to mitochondrial heteroplasmy. While we were unable to conduct this experiment, we compared genetic estimates of reproductively active adult worms with direct counts of adult worms from nodules extracted from 9 Ghanaian participants. We did not find a significant correlation between adult worm burden estimates based on mf genetic data and the fertile worm counts from nodules, although the genetic estimates were equal to or higher than the nodule worm counts in most cases. Accurately quantifying adult worm burden via nodulectomy can be difficult due to the presence of hidden nodules that cannot be palpated, as they may reside in deeper subcutaneous layers or within the body cavity [11–13]. Additionally, microfilariae can survive for approximately 18–24 months after being released by the female worm [11], making it possible that some of the genotyped mf in this study originated from infertile or deceased females that had stopped reproducing prior to nodulectomy. In 2 of the 9 participants, genetic estimates of adult males were possible despite no adult male worms being identified in the examined nodules. Since males migrate between nodules [14], they may not have been present in any nodules at the time the nodulectomies were performed. One limitation of our data is that we do not have pre- and post-treatment samples, and thus could not explicitly test some of the alternative hypotheses for ongoing transmission of O. volvulus in Ghana. However, we have recently shown that nuclear genome-based analysis of mf can effectively distinguish between recrudescence and reinfection in lymphatic filariasis [35], and we propose that similar approaches could be applied to O. volvulus.

Our study has significantly expanded the number of nuclear genomes available for population genomic analysis, increasing the dataset by more than 300 genomes, compared to the ~ 30 previously sequenced at comparable depth and coverage [36,37,39]. This new nuclear genome data, derived from individual single mf, enabled the first accurate comparison of genetic variation between sex chromosomes and autosomes in O. volvulus, without the confounding effects of embryonic DNA that can compromise the genotyping accuracy of maternal DNA in adult worm sequencing. Using 28 unrelated female mf samples (≥ third-degree kinship) from Ghana, we analyzed patterns of nucleotide diversity (π) and LD across the genome. Our genome-wide estimate of nucleotide diversity in the nuclear genome (π = 2 ×ばつ 10^-3) was an order of magnitude lower than that of the mitochondrial genome (π = 3 ×ばつ 10^-2) and lower than our previous global estimate (π = 4 ×ばつ 10^-3) [36], yet notably higher than values reported for other vector-borne filarial parasites, Wuchereria bancrofti (π = 2 ×ばつ 10^-4) [49] and Dirofilaria immitis (π = 4 ×ばつ 10^-5 to 7 ×ばつ 10^-4) [50]. Negative Tajima’s D values were observed across both the nuclear and mitochondrial genomes, indicating an excess of low-frequency polymorphisms relative to expectations under neutrality. This pattern may reflect recent population expansion following a bottleneck. Our findings revealed lower than expected sequence diversity and extended LD in the non-PAR region of the X chromosome, compared to the PAR and autosomes. Comparative genomic analyses of filarial nematode species indicate that a recent X-autosome fusion event led to neo-X/Y formation in the last common ancestor of Dirofilaria and Onchocerca [51,52]. This fusion likely altered recombination patterns, suppressing it near the fusion point and resulting in a loss of genetic diversity [53,54]. Low X-linked genetic diversity appears to be a hallmark of filarial species that have undergone neo-X/Y formation [51].

Patterns of LD decay provide valuable insights into historical recombination events. LD is influenced by both the recombination rate and the number of generations that have undergone recombination [55]. Our evidence suggests that the X chromosome in O. volvulus likely experiences a lower recombination rate than the rest of the genome, making it more vulnerable to Muller’s Ratchet [56,57], a process that leads to the stochastic accumulation of mildly deleterious mutations over time. Additionally, recessive variants under positive or purifying selection are expected to go to fixation more rapidly on the X chromosome due to hemizygosity in males, who carry only one copy of the X chromosome. This makes genes in the non-PAR more "exposed" to selection [58], potentially reducing genetic variation across large portions of the X chromosome. Given that genetic diversity is a key determinant of a population’s ability to adapt to changing environmental conditions, including exposure to anthelmintics or different vector species, the reduced genetic diversity on the X chromosome, which accounts for ~28% of the O. volvulus genome, may have significant evolutionary consequences.

In conclusion, our findings suggest that fine-scale genetic structure in O. volvulus populations can be detected using mf whole-genome nuclear data, offering greater resolution than previous studies that used mitochondrial data alone. Additionally, we applied sibship reconstruction with autosomal and sex chromosomal SNPs to estimate the minimum number of reproductively active adult females and males in individual hosts. This work lays the groundwork for developing a genetic approach that could provide invaluable information for the MDA program. Furthermore, our whole-genome data enabled a detailed analysis of genome-wide genetic variation, confirming lower-than-expected genetic diversity on the X chromosome. This study contributes to the application of modern population genomics tools in support of onchocerciasis elimination efforts.

Methods

Supporting information

Acknowledgments

We thank the late Fatorma K. Bolay for his support during sample collection in Liberia. We thank Kirsten Grant for assistance in the lab, all the study participants from Ghana, Liberia, and the Democratic Republic of the Congo for providing samples, and the McDonnell Genome Institute at Washington University School of Medicine for generating the sequence data. For sample contribution, we thank the MDGH-MOX-3001 and MDGH-MOX-3002 programs in the DRC that were facilitated by the European and Developing Countries Clinical Trials Partnership (https://www.edctp.org; RIA2017NCT-1843), Medicines Development for Global Health (https://www.medicinesdevelopment.com), and the Luxembourg National Research Fund (https://www.fnr.lu; Inter/Vaillant/2018).

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