Phenotypic Evidence of Emerging Ivermectin Resistance in Onchocerca volvulus Mike Y. Osei-Atweneboana 1,5 , Kwablah Awadzi 2 , Simon K. Attah 2 , Daniel A. Boakye 3 , John O. Gyapong 4,6 , Roger K. Prichard 1 * 1 Institute of Parasitology, McGill University, Montreal, Canada, 2 Onchocerciasis Chemotherapy Research Centre, Hohoe, Ghana, 3 Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana, 4 Health Research Unit, Ghana Health Service, Accra, Ghana, 5 Council for Scientific and Industrial Research, Accra, Ghana, 6 Ochocerciasis Control Programme of Ghana, Accra, Ghana Abstract Background: Ivermectin (IVM) has been used in Ghana for over two decades for onchocerciasis control. In recent years there have been reports of persistent microfilaridermias despite multiple treatments. This has necessitated a reexamination of its microfilaricidal and suppressive effects on reproduction in the adult female Onchocerca volvulus. In an initial study, we demonstrated the continued potent microfilaricidal effect of IVM. However, we also found communities in which the skin microfilarial repopulation rates at days 90 and 180 were much higher than expected. In this follow up study we have investigated the reproductive response of female worms to multiple treatments with IVM. Methods and Findings: The parasitological responses to IVM in two hundred and sixty-eight microfilaridermic subjects from nine communities that had received 10 to 19 annual doses of IVM treatment and one pre-study IVM-naı ¨ve community were followed. Skin snips were taken 364 days after the initial IVM treatment during the study to determine the microfilaria (mf) recovery rate. Nodules were excised and skin snips taken 90 days following a second study IVM treatment. Nodule and worm density and the reproductive status of female worms were determined. On the basis of skin mf repopulation and skin mf recovery rates we defined three categories of response—good, intermediate and poor—and also determined that approximately 25% of subjects in the study carried adult female worms that responded suboptimally to IVM. Stratification of the female worms by morphological age and microfilarial content showed that almost 90% of the worms were older or middle aged and that most of the mf were produced by the middle aged and older worms previously exposed to multiple treatments with little contribution from young worms derived from ongoing transmission. Conclusions: The results confirm that in some communities adult female worms were non-responsive or resistant to the anti-fecundity effects of multiple treatments with IVM. A scheme of the varied responses of the adult female worm to multiple treatments is proposed. Citation: Osei-Atweneboana MY, Awadzi K, Attah SK, Boakye DA, Gyapong JO, et al. (2011) Phenotypic Evidence of Emerging Ivermectin Resistance in Onchocerca volvulus. PLoS Negl Trop Dis 5(3): e998. doi:10.1371/journal.pntd.0000998 Editor: Sara Lustigman, New York Blood Center, United States of America Received April 12, 2010; Accepted March 3, 2011; Published March 29, 2011 Copyright: ß 2011 Osei-Atweneboana 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. Funding: This study received support from the Canadian Institutes for Health Research (http://www.cihr-irsc.gc.ca/e/193.html), grant No. 94593, the Ghana Government and the Centre for Host-Parasite Interactions (http://www.mcgill.ca/chpi/), McGill University, Canada. The funders had no role in 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. * E-mail: roger.prichard@mcgill.ca Introduction Ivermectin (Mectizan) has been in operational use for the control of human onchocerciasis in the former Onchocerciasis Control Programme in West Africa (OCP) areas since 1987 and is still the drug of choice for the African Programme for Onchocerciasis Control (APOC) and the Onchocerciasis Elimi- nation Programme for the Americas (OEPA). A single dose of ivermectin rapidly kills skin microfilariae (mf) and inhibits microfilarial release by adult female worms, possibly because of paralysis of the lower uterus [1]. These effects result in a rapid decline in skin microfilarial counts, an accumulation of degener- ating intra-uterine microfilariae and a long-lasting suppression of Onchocerca volvulus microfilaridermias [2]. However, the inhibition of mf release is reversible by six months resulting in the reappearance of microfilariae in the skin (repopulation) and after a further six months a restitution of a proportion of the initial load (recovery) [2]. A proportion of ivermectin exposed female worms do not resume reproductive activity even after one year [1] and this can persist for up to 18 months after treatment [3]. The prolonged microfilarial suppressant effect of ivermectin has beneficial effects on morbidity and on parasite transmission. Several studies [1,4,5,6,7,8] have demonstrated that multiple treatments with ivermectin have marked suppressive effects on embryogenesis consistent with some cumulative effect of ivermec- tin treatments on microfilarial production. Quantitative estimates have ranged from an irreversible decline in microfilarial production of ,30% after five annual treatments [9], a reduction in the productivity index of 90% or more after 10 six-monthly doses over 6 years [10], to arrest of development at the single cell stage after four or five six-monthly doses [11]. Recently, the cumulative effect of ivermectin on microfilarial production by O. www.plosntds.org 1 March 2011 | Volume 5 | Issue 3 | e998