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Skin infections by the ectoparasitic mite Sarcoptes scabiei are a preventable source of morbidity worldwide. While scabies affects all socioeconomic sectors, it is especially prominent in the developing world where crowding, poor hygiene, and limited access to basic health care are commonplace. Mass eradication efforts of this parasite have historically been hampered by delivery and compliance issues surrounding topical standards of care. There have been advances in eradication over the last decade due to the expanded use of oral Ivermectin for the treatment and prevention of ectoparasites such as scabies. Previous research focused on various treatment aspects of the disease, yet most identified the need for basic scabies health education to accompany future studies to improve program sustainability and the overall health literacy of target populations. The Virginia College of Osteopathic Medicine sponsors a public health clinic in Veron, Dominican Republic that reports a high prevalence of scabies infections among its general patient population. The lack of any means of direct measurement, proper intervention, or control raises concerns that long-term infection may lead to multiple other secondary disease sequelae. The Veron Scabies Eradication and Education Program was designed to address this deficiency and build on previous studies related to this problem by using a novel treatment and education protocol. The purpose of this research was to identify, treat, and prevent primary and secondary health problems due to long-term scabies infection in the community of Barrio Nuevo, Veron, Dominican Republic in order to provide long-term sustainable eradication through a researcher-designed health education program and medical service protocol. Participants continued typical daily life while interventions and education were monitored to detect change over a 9-month timeline using selected measurement intervals to test multiple scabies disease and knowledge objectives and related hypotheses. Following this basic protocol, the following scabies markers were evaluated at baseline: subject demographics, scabies diagnosis and treatment history, baseline treatments and reported side effects, and scabies education pre-test results. The following scabies markers were also assessed at baseline and reassessed at 2 weeks, 1 month, 2 months, 8 months, and 9 months: risk and prevention behaviors, symptomatology, skin exam findings, and scabies education post-test results. There was a statistically significant post-intervention improvement in scabies markers when compared to pre-intervention values (p<0.05). This study demonstrated that a community scabies program involving large-scale treatment and education can provide rapid and long lasting improvements to the health of a highly endemic population. Community-wide scabies eradication is possible with the appropriate level of structure and support using low cost medication available to the health care system that serves Veron, Dominican Republic. Given the efficacy and safety profile of Ivermectin demonstrated in this study and substantiated by others, it is recommended that the Dominican Republic Ministry of Health consider adopting Ivermectin as the standard of care for scabies treatments and enforce an existing formal ban on Lindane products. The protocol from this research should be considered for adoption to provide a sustainable, practical, self-sufficient model for improved health outcomes, health behaviors, and health literacy. Future studies should replicate this research to determine validation in other settings, cultures and situations, build on the findings by exploring additional variables related to environmental risk factors, and continue to develop interventions that promote health education and enhance clinical practices. / Ph. D.

Níveis não aceitáveis de resíduos de ivermectina (IVM), uma droga anti-parasitária amplamente utilizada no Brasil para controle de endectoparasitas, pode estar presente no leite para consumo humano se administrada incorretamente. O nível máximo de resíduos no leite para este composto é de 10 ng mL-1 e sua presença tem sido comprovada em pesquisas realizadas por orgãos reguladores. Com o conhecimento deste problema, um método para a detecção de ivermectina em amostras de leite foi desenvolvido utilizando a extração líquido-líquido com base em acetonitrila e hexano, seguida por derivatização com 1-metilimidazol (MI), trietilamina (TEA), ácido anidro trifluoroacético (TFAA) e ácido trifluoroacético (TFA) e cromatografia líquida com detecção fluorescente (LC-FL) para a análise. Além disso, o método proposto foi testado de acordo com parâmetros de validação estabelecidos pela ANVISA. Parâmetros, tais como seletividade, linearidade (R² 0,98), precisão (coeficiente de variação entre 0,6-19%), recuperação (90-95%) e robustez foram avaliados durante o processo de validação. Subsequentemente, foi testado em amostras de leite de vacas tratadas com uma formulação comercial de ivermectina a 1%. O método aplicado em amostras de campo, provou possuir um perfil de quantificação e de confirmação para o método concebido no presente estudo. / Non-acceptable residue levels of ivermectin (IVM), an anti-parasitic drug widely employed in Brazil for control of endectoparasites, could be present in milk for human consumption if improperly administered. The maximum residue level in milk for this compound is 10 ng mL-1 and its presence has been proved from previous government surveys. With this rising subject, a method for the detection of ivermectin in milk samples was developed using liquid-liquid extraction based in acetonitrile and hexane, followed by derivatization with 1-methylimidazole (MI), triethylamine (TEA), anhydrous trifluoacetic acid (TFAA) and trifluoracetic acid (TFA) and liquid chromatography coupled to fluorescent detection (LC-FL) for the analysis. Moreover, the proposed method was tested according to validation parameters estabilished by ANVISA. Parameters, such as selectivity, linearity (R² 0.98), precision (RSD values between 0.6 19%), recovery (90-95%) and robustness were evaluated during the validation process. Subsequently it was tested in milk samples from cows treated with a commercial ivermectin formulation at 1%. The method applied to field samples, proved a quantifiable and confirmatory profile for the method designed in this study.

Purpose: Ivermectin (IVM) is a lipophilic BCS-II compound (molecular weight=875 g/mole, LogP=3.22, intrinsic solubility=700 ug/L). IVM is used as antiparasitic drug in both humans and animals. IVM is known to have a half-life of 12-56 hours in humans. Strongyloidiasis is a chronic parasitic infection of humans caused by Strongyloides stercoralis, with an estimated 30-100 million people infected worldwide. Infection may be severe and even life-threatening in cases of immunodeficiency. Patients with disseminated strongyloidiasis are usually bedridden hospitalized patients that show symptoms such as paralytic ileus and reduced plasma albumin and cholesterol. Oral IVM is the only FDA-approved treatment but may not be effective in patients with disseminated disease. Veterinary subcutaneous formulations have been used in severe infections. We hypothesized that IVM PK in patients with disseminated strongyloidiasis can be predicted using PBPK model originally built and refined in healthy human and animal species. This hypothesis was tested and shown to be valid. Methods:A systematic method was used to build and refine different parts of the PBPK model. The process involved construction of models, parameterization of these models, evaluation of the effect of uncertainty in model parameters on model prediction via local and global sensitivity analyses and finally, refinement of model predictions. Two disposition models that differ in the rate limiting step in drug distribution were constructed and include perfusion-limited and permeability-limited distribution models. The ability of each model to predict IVM disposition was evaluated using plasma PK data in rat after intra-arterial dosing and in dog after intravenous bolus dosing. Then the disposition model was scaled to humans and an oral input model was constructed as a modification on the well-known ACAT model. The oral input model was coupled with the disposition model and used to predict IVM plasma concentration-time profile in healthy fasted human subject after oral dosing. Two subcutaneous (SQ) input models were constructed and used to evaluate the effect of IVM precipitation at the injection site. Plasma PK data in dog after SQ dosing was used to refine the constructed SQ input models. The refined disposition, oral input and SQ input physiologically-based models were used to predict IVM PK in patients with disseminated strongyloidiasis after a complex dosing regimen. The physiological parameters of the model were modified to account for the effect of the disease-induced pathophysiological changes on the body physiology and hence on the drug PK. Plasma PK data from hospitalized subjects with disseminated strongylidiasis was used in this part. Results and conclusions:The disposition model with assumption of permeability-limited distribution was more capable of describing IVM disposition in rat after intra-arterial dosing compared to when perfusion-limited distribution was assumed. The model predicted that hepatic clearance is the most impactful parameter on model-predicted plasma concentration of the drug. Also, IVM was shown to have low hepatic extraction ratio along with high binding in plasma and large volume of distribution, which collectively may explain the long half-life in the plasma of 63 hours in rat after intra-arterial dosing. The oral input model predicted that the oral input is limited by drug dissolution in the GI lumen and that a very small fraction of oral tablet dose (0.03) is available in the systemic circulation in healthy fasted human subjects. Both of the studied SQ input models predicted that majority of IVM absorption after SQ dosing is via the lymphatic route and that drug precipitation at the injection site can further slowdown the drug absorption after SQ administration. The PBPK model was able achieve the main goal of this research which is to predict IVM pharmacokinetics in patients with disseminated strongyloidiasis after a complex dosing regimen of multiple oral and SQ dosing. This was achieved by modifying the most impactful physiological parameters of the model affected by the disease state and that are related to drug binding in the plasma (fraction unbound), the GI motility (gastric emptying rate) and the lymphatic flow rate. Based on our analysis, we recommend measurement of plasma IVM concentrations early after initiation of therapy to exclude treatment failure due to reduced oral and/or SQ absorption. Also, we recommend measurement of plasma lipoprotein levels and their composition in these patients to differentiate between low total plasma concentrations due to low binding plasma as opposed to low drug input. Finally, interventional procedures that enhance lymphatic flow rate to site of SQ injection are recommended to enhance SQ absorption.

Anthelmintic treatment of livestock is an important aspect of the control of gastrointestinal parasites. Resistance to anthelmintics is common, and an understanding of resistance requires knowledge of an anthelmintic's mode(s) of action and mechanism(s) of resistance. The parasitic nematode, Haemonchus contortus, has developed resistance to benzimidazoles and avermectins/milbemycins. Proposed mechanisms of resistance are here supported by genetic changes observed in genes whose protein products are believed to interact with these anthelmintics. Statistically significant differences in allele frequencies were observed between untreated and ivermectin- and moxidectin-treated strains in a gene encoding a putative glutamate-gated chloride channel alpha subunit, a proposed target of avermectins/milbemycins. One allele appeared to be associated with resistance. Similar changes in allele frequencies in the same strains occurred in a gene encoding a subunit of a gamma-aminobutyric acid receptor. Significant differences in allele frequencies of a gene encoding a P-glycoprotein were found in strains of H. contortus treated with ivermectin and moxidectin compared to derived, untreated strains. In all treated strains, one allele appeared to be associated with resistance. Similarly, allele frequencies of this gene were significantly different between a cambendazole-treated strain and its derived, untreated strain. These results implicate glutamate-gated chloride channels and gamma-aminobutyric acid receptors in mechanisms of resistance to avermectins/milbemycins and implicate P-glycoprotein in a mechanism of resistance to avermectins/milbemycins and benzimidazoles in H. contortus.

The anti-nematodal drug ivermectin hyperactivates invertebrate-specific glutamate-gated chloride channels (GluCls) causing pharyngeal paralysis and a cessation of feeding and growth. I find that for C. elegans even brief exposure to ivermectin can lead to irreversible pharyngeal paralysis. Ivermectin induces heterogeneous vacuolation in the pharynx that appears slowly and accumulates over several days. This vacuolation is almost completely rescued by a mutation in avr-15, which codes for the alpha-subunit of pharyngeal GluCls. The vacuoles stain strongly with Lysotracker Red and are therefore likely to be acidic compartments of the endosomal-lysosomal system. Examination of mutants defective for endocytosis (rme-1, rme-8, and cup-5) uncovers the presence of acidic vacuoles identical in appearance to ivermectin-induced vacuoles. Further, RME-1, a marker for recycling endosomes, is shown to redistribute soon after ivermectin exposure. Examination of the effects of ivermectin on extrapharyngeal neurons expressing ectopic avr-15 reveals an apoptotic phenotype that is shown to be ced-independent.

Resistance to the avermectin/milbemycin class of anthelmintics in nematodes has become a serious problem worldwide due to their unrestricted usage. Resistance to these compounds is attributed to the over-expression of the transport protein, P-glycoprotein (P-gp). P-gp acts by pumping drug molecules out from the cell or organism, P-gp efflux activity can be blocked using multidrug resistance (MDR) modulators associated with chemotherapy to enhance their therapeutic effect. A series of experiments was undertaken to determine if the association of the anthelmintics, ivermectin (IVM) and moxidectin (MOX), and MDR modulators would increase the anthelmintics' efficacy against resistant parasites. Using an in vitro migration assay, IVM and MOX in the presence or absence of verapamil (VRP), CL347,099 and cyclosporin A (CyA) were used against IVM- and MOX-selected strains of H. contortus. The modulators alone had no effect on reducing the number of migrating larvae, IVM and MOX had a significant increase in efficacy of 52.7 and 58,3% respectively, when used in association with VRP, above that obtained with the anthelmintics alone. CL347,099 was also able to significantly increase the IVM and MOX efficacy by 24.2 and 38.9%, respectively. The effect of IVM and MOX in combination with VRP and CL347,099 was determined in jirds infected with selected strains of H. contortus. The combinations of VRP with either IVM or MOX significantly reduced worm counts of the selected strains compared with the untreated controls, whereas IVM or MOX alone did not. CL347,099 plus MOX combination was significantly more efficacious than moxidectin alone against the selected strains. To evaluate the effect of VRP on the pharmacokinetic behaviour of the anthelmintics IVM and MOX, the drug combination was given to sheep. The IVM plus VRP treatment resulted in an increase of the pharmacokinetic parameters of IVM. The peak concentration (83%) and area under the curve (54%) were significantly differen

Anthelmintics, antiparasitic agents, have been developed as a main weapon to control parasitic nematodes of domestic ruminants. Unfortunately, the intensive use of anthelmintics leads to the development of drug resistance in parasite populations. Anthelmintic resistance has compromised the control of nematode parasites and has become a major problem in many countries of the world. Resistance to the newest anthelmintics such as ivermectin (IVM) and related anthelmintics in Haemonchus contortus in sheep has been developing rapidly in recent years. The development of drug resistance is an evolutionary process that leads to genetic changes in parasite populations in response to drug exposure. However, the mechanism of ivermectin resistance in nematode parasites is unknown. P-glycoprotein (Pgp) has been well documented in mammalian cells as a membrane transporter by actively extruding a variety of structurally and functionally unrelated hydrophobic cytotoxic drugs out of the cell. This study was to determine whether there is an association between specific alleles at the Pgp locus and IVM or moxidectin (MOX) selection in H. contortus, by investigating the genetic variation of the Pgp homologue in unselected and IVM- and MOX-selected strains of H. contortus. (Abstract shortened by UMI.)

Onchocerca volvulus, a filarial nematode, is the causative agent of onchocerciasis. / O. volvulus is a human parasite with no animal model host and is endemic in the tropics. O. volvulus material is scarce and must be conserved as part of the Onchocerciasis Control Program. A genomic library was constructed to provide a substantial source of renewable genetic material, in place of original parasite DNA. / Currently there is only one glutamate-gated chloride channel that has been sequenced from O. volvulus, but this has not yet been characterized. This GluClx partial cDNA sequence isolated by Cully et al., 1997, may be found in GenBank, accession number U59745. Specific primers were designed to amplify this gene from the genomic library. A fragment of this gene was isolated but the primers were non-specific, amplifying genes in addition to GluClx. / A motif is a short recognition sequence within a protein that may allow the modification of the protein. The cysteine loop in the N-terminal of all the ligand-gated ion channels is interesting because it contains the neurotransmitter-gated ion channel signature sequence. (Abstract shortened by UMI.)

Anthelmintic resistance is a major problem in livestock, and while it has been slower to emerge in cattle, there are reports of its occurrence. Three broad-spectrum anthelmintics are available for use, and one mechanism of resistance that is common to all is target site alteration. Glutamate-gated chloride channels (GluCls) are an important target for macrocyclic lactone anthelmintics (MLs), while beta-tubulin represents the benzimidazole (BZ) target. The objectives of this thesis were to determine whether GluCls are involved in ML resistance in the cattle parasite Cooperia oncophora , and whether beta-tubulin is involved in BZ and ML resistance. Two isolates of C. oncophora were used. In a fecal egg-count reduction test, ivermectin was found to be 100% effective against one isolate (IVS), and only 77.8% effective against the second isolate (IVR). Two full-length GluCl cDNAs, encoding GluClalpha3 and beta subunits, were cloned. These subunits share high sequence identity with similar GluCl subunits from Haemonchus contortus and Caenorhabditis elegans. Genetic variability analysis of the two genes showed significant differences in allele frequencies between IVS and IVR worms at the GluClalpha3 gene, but not the GluClbeta gene, suggesting that the GluClalpha3 gene is involved in ivermectin resistance. Sequencing of full-length GluCl subunit cDNAs from IVS and IVR worms revealed the presence of mutations in the N-terminal domains. Mutations in the GluClalpha3 caused modest but significant reductions in glutamate, ivermectin and moxidectin sensitivity, while mutations in the GluClbeta abolished glutamate sensitivity. Of the three mutations identified in the IVR GluClalpha3, the L256F mutation accounted for the difference in glutamate and ivermectin response between IVS and IVR GluClalpha3 channels. Two beta-tubulin isotypes cloned from C. oncophora were found to share a high homology with beta-tubulin isotypes from other trichostrongylids. Gen