Genome-wide analysis of ivermectin response by Onchocerca volvulus reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity

Treatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana-exposed to more than a decade of regular ivermectin treatment-have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread.Pooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR.This study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations

A Research Agenda for Helminth Diseases of Humans: Towards Control and Elimination

Human helminthiases are of considerable public health importance in sub-Saharan Africa, Asia, and Latin America. The acknowledgement of the disease burden due to helminth infections, the availability of donated or affordable drugs that are mostly safe and moderately efficacious, and the implementation of viable mass drug administration (MDA) interventions have prompted the establishment of various large-scale control and elimination programmes. These programmes have benefited from improved epidemiological mapping of the infections, better understanding of the scope and limitations of currently available diagnostics and of the relationship between infection and morbidity, feasibility of community-directed or school-based interventions, and advances in the design of monitoring and evaluation (M&E) protocols. Considerable success has been achieved in reducing morbidity or suppressing transmission in a number of settings, whilst challenges remain in many others. Some of the obstacles include the lack of diagnostic tools appropriate to the changing requirements of ongoing interventions and elimination settings; the reliance on a handful of drugs about which not enough is known regarding modes of action, modes of resistance, and optimal dosage singly or in combination; the difficulties in sustaining adequate coverage and compliance in prolonged and/or integrated programmes; an incomplete understanding of the social, behavioural, and environmental determinants of infection; and last, but not least, very little investment in research and development (R&D). The Disease Reference Group on Helminth Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR), was given the mandate to undertake a comprehensive review of recent advances in helminthiases research, identify research gaps, and rank priorities for an R&D agenda for the control and elimination of these infections. This review presents the processes undertaken to identify and rank ten top research priorities; discusses the implications of realising these priorities in terms of their potential for improving global health and achieving the Millennium Development Goals (MDGs); outlines salient research funding needs; and introduces the series of reviews that follow in this PLoS Neglected Tropical Diseases collection, “A Research Agenda for Helminth Diseases of Humans.

The transcriptional response of Caenorhabditis elegans to ivermectin exposure identifies novel genes involved in the response to reduced food intake

We have examined the transcriptional response of Caenorhabditis elegans following exposure to the anthelmintic drug ivermectin (IVM) using whole genome microarrays and real-time QPCR. Our original aim was to identify candidate molecules involved in IVM metabolism and/or excretion. For this reason the IVM tolerant strain, DA1316, was used to minimise transcriptomic changes related to the phenotype of drug exposure. However, unlike equivalent work with benzimidazole drugs, very few of the induced genes were members of xenobiotic metabolising enzyme families. Instead, the transcriptional response was dominated by genes associated with fat mobilization and fatty acid metabolism including catalase, esterase, and fatty acid CoA synthetase genes. This is consistent with the reduction in pharyngeal pumping, and consequential reduction in food intake, upon exposure of DA1316 worms to IVM. Genes with the highest fold change in response to IVM exposure, cyp-37B1, mtl-1 and scl-2, were comparably up-regulated in response to short–term food withdrawal (4 hr) independent of IVM exposure, and GFP reporter constructs confirm their expression in tissues associated with fat storage (intestine and hypodermis). These experiments have serendipitously identified novel genes involved in an early response of C. elegans to reduced food intake and may provide insight into similar processes in higher organisms

Onchocerciasis transmission in Ghana: Persistence under different control strategies and the role of the simuliid vectors

Background: The World Health Organization (WHO) aims at eliminating onchocerciasis by 2020 in selected African countries. Current control focuses on community-directed treatment with ivermectin (CDTI). In Ghana, persistent transmission has been reported despite long-term control. We present spatial and temporal patterns of onchocerciasis transmission in relation to ivermectin treatment history. Methodology/Principal Findings: Host-seeking and ovipositing blackflies were collected from seven villages in four regions of Ghana with 3–24 years of CDTI at the time of sampling. A total of 16,443 flies was analysed for infection; 5,812 (35.3%) were dissected for parity (26.9% parous). Heads and thoraces of 12,196 flies were dissected for Onchocerca spp. and DNA from 11,122 abdomens was amplified using Onchocerca primers. A total of 463 larvae (0.03 larvae/fly) from 97 (0.6%) infected and 62 (0.4%) infective flies was recorded; 258 abdomens (2.3%) were positive for Onchocerca DNA. Infections (all were O. volvulus) were more likely to be detected in ovipositing flies. Transmission occurred, mostly in the wet season, at Gyankobaa and Bosomase, with transmission potentials of, respectively, 86 and 422 L3/person/month after 3 and 6 years of CDTI. The numbers of L3/1,000 parous flies at these villages were over 100times the WHO threshold of one L3/1,000 for transmission control. Vector species influenced transmission parameters. At Asubende, the number of L3/1,000 ovipositing flies (1.4, 95% CI = 0–4) also just exceeded the threshold despite extensive vector control and 24 years of ivermectin distribution, but there were no infective larvae in host-seeking flies. Conclusions/Significance: Despite repeated ivermectin treatment, evidence of O. volvulus transmission was documented in all seven villages and above the WHO threshold in two. Vector species influences transmission through biting and parous rates and vector competence, and should be included in transmission models. Oviposition traps could augment vector collector methods for monitoring and surveillance

A Research Agenda for Helminth Diseases of Humans: Health Research and Capacity Building in Disease-Endemic Countries for Helminthiases Control

Capacity building in health research generally, and helminthiasis research particularly, is pivotal to the implementation of the research and development agenda for the control and elimination of human helminthiases that has been proposed thematically in the preceding reviews of this collection. Since helminth infections affect human populations particularly in marginalised and low-income regions of the world, they belong to the group of poverty-related infectious diseases, and their alleviation through research, policy, and practice is a sine qua non condition for the achievement of the United Nations Millennium Development Goals. Current efforts supporting research capacity building specifically for the control of helminthiases have been devised and funded, almost in their entirety, by international donor agencies, major funding bodies, and academic institutions from the developed world, contributing to the creation of (not always equitable) North–South “partnerships”. There is an urgent need to shift this paradigm in disease-endemic countries (DECs) by refocusing political will, and harnessing unshakeable commitment by the countries' governments, towards health research and capacity building policies to ensure long-term investment in combating and sustaining the control and eventual elimination of infectious diseases of poverty. The Disease Reference Group on Helminth Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR), was given the mandate to review helminthiases research and identify research priorities and gaps. This paper discusses the challenges confronting capacity building for parasitic disease research in DECs, describes current capacity building strategies with particular reference to neglected tropical diseases and human helminthiases, and outlines recommendations to redress the balance of alliances and partnerships for health research between the developed countries of the “North” and the developing countries of the “South”. We argue that investing in South–South collaborative research policies and capacity is as important as their North–South counterparts and is essential for scaled-up and improved control of helminthic diseases and ultimately for regional elimination

Metabolomics-Based Discovery of Diagnostic Biomarkers for Onchocerciasis

Onchocerciasis, caused by the filarial parasite Onchocerca volvulus, afflicts millions of people, causing such debilitating symptoms as blindness and acute dermatitis. There are no accurate, sensitive means of diagnosing O. volvulus infection. Clinical diagnostics are desperately needed in order to achieve the goals of controlling and eliminating onchocerciasis and neglected tropical diseases in general. In this study, a metabolomics approach is introduced for the discovery of small molecule biomarkers that can be used to diagnose O. volvulus infection. Blood samples from O. volvulus infected and uninfected individuals from different geographic regions were compared using liquid chromatography separation and mass spectrometry identification. Thousands of chromatographic mass features were statistically compared to discover 14 mass features that were significantly different between infected and uninfected individuals. Multivariate statistical analysis and machine learning algorithms demonstrated how these biomarkers could be used to differentiate between infected and uninfected individuals and indicate that the diagnostic may even be sensitive enough to assess the viability of worms. This study suggests a future potential of these biomarkers for use in a field-based onchocerciasis diagnostic and how such an approach could be expanded for the development of diagnostics for other neglected tropical diseases

Potential effects of warmer worms and vectors on onchocerciasis transmission in West Africa

Development times of eggs, larvae and pupae of vectors of onchocerciasis (Simulium spp.) and of Onchocerca volvulus larvae within the adult females of the vectors decrease with increasing temperature. At and above 25C,the parasite could reach its infective stage in less than 7 days when vectors could transmit after only two gonotrophic cycles. After incorporating exponential functions for vector development into a novel blackfly population model, it was predicted that fly numbers in Liberia and Ghana would peak at air temperatures of 29C and 34C, about 3C and 7C above current monthly averages, respectively; parous rates of forest flies (Liberia) would peak at 298C and of savannah flies (Ghana) at 308C. Small temperature increases (less than 28C) might lead to changes in geographical distributions of different vector taxa. When the new model was linked to an existing framework for the population dynamics of onchocerciasis in humans and vectors, transmission rates and worm loads were projected to increase with temperature to at least 338C. By contrast, analyses of field data on forest flies in Liberia and savannah flies in Ghana, in relation to regional climate change predictions, suggested, on the basis of simple regressions, that 13–41% decreases in fly numbers would be expected between the present and before 2040. Further research is needed to reconcile these conflicting conclusions

Macrofilaricidal Activity after Doxycycline Only Treatment of Onchocerca volvulus in an Area of Loa loa Co-Endemicity: A Randomized Controlled Trial

The control of onchocerciasis in Africa relies on the sustained delivery of ivermectin. In certain areas, annual treatments delivered with high population coverage for at least 15–17 years can break transmission. In other endemic settings this strategy alone is thought to be insufficient to eradicate the disease. One of the major limitations occurs in areas that are co-endemic with another filarial infection caused by Loa loa, due to the risk of a rare severe adverse event associated with the rapid killing of L. loa microfilariae in heavily parasitized individuals. There are also concerns over recent evidence of reduced efficacy of ivermectin and the possible development of resistance. An alternative approach is to target the Wolbachia bacterial endosymbionts of Onchocerca volvulus with the antibiotic, doxycycline. In an area of Cameroon co-endemic for onchocerciasis and loiasis we conducted a trial comparing doxycycline with or without ivermectin treatment to ivermectin treatment alone. A six-week course of doxycycline delivers macrofilaricidal and sterilizing activities, which is not dependent upon co-administration of ivermectin. Doxycycline is well tolerated in patients co-infected with moderate intensities of L. loa microfilariae. The trial indicates that anti-wolbachial therapy is a feasible alternative to ivermectin in communities co-endemic for onchocerciasis and loiasis

Mining Predicted Essential Genes of Brugia malayi for Nematode Drug Targets

We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression

Glutamate-Gated Chloride Channels of Haemonchus contortus Restore Drug Sensitivity to Ivermectin Resistant Caenorhabditis elegans

Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance