Characterization of Onchocerca Volvulus Response to Ivermectin Treatment And Identification of Single Nucleotide Polymorphisms Associated With These Responses in Some Onchocerciasis Endemic R

ABSTRACT

In Ghana, onchocerciasis control with mass ivermectin treatment began in 1987. Despite over two decades of interventions, the disease remains persistent with reports of sub-optimal/poor parasite responses to the drug. Some treated patients are observed with higher microfilarial repopulation rates in skin than expected, an indication of sub-optimal response. Although ivermectin is still effective in reducing microfilaridermias, it is uncertain if its embryostatic effect has been compromised. This thesis was to assess the impact of the first 3 years of biannual treatment strategy in Ghana and quantify responses to standard dose of ivermectin in hosts’ skin, assess the drug’s effect on the reproductive capacities of adult female worms and explore any genetic changes in beta-tubulin (β-tub) and P-glycoprotein (P-gp) genes that are believed to be associated with poor response phenotype. The study was carried out in 10 sentinel communities which had received between 15 and 24 years of annual treatment. A community-wide skin snipping was performed on 956 consenting adults aged ≥20 years to assess the community prevalence and intensity of microfilariae. A cohort of 217 participants who were microfilaria positive and/or had palpable nodules at baseline were followed up over the first two rounds of biannual treatment to estimate the rates of microfilarial repopulation. Nodulectomies were performed on consenting participants three months after the third round of treatment. Adult worms (male and female) were isolated from nodules using the collagenase technique. Embryogram analyses were performed and adult female worms classified into three response groups (good, intermediate and poor). DNA was extracted from 60 worms which accurately fitted the response classifications. Polymerase Chain Reaction (PCR) amplifications were performed using specific primers for one region within β-tub and 6 regions within P-gp genes. The amplified products were sequenced and analysed for single nucleotide polymorphisms (SNPs) associated with these responses. The biannual treatment substantially reduced infection intensities in most communities, although infections were detected in all communities even after 4 or 5 rounds of biannual treatments. Asubende, Kyingakrom and New-Longoro communities were identified (all having been previously recognized as responding sub-optimally to ivermectin) with statistically significantly high microfilarial repopulation rates. A total of 225 nodules were excised from 106 participants with an average of about 2 female worms and 1 male worm per nodule. A significantly higher number of female worms (72%) were observed without normal/viable microfilariae (P < 0.0001). There were no clear associations between the years of annual ivermectin treatment prior to biannual and microfilarial repopulation rates or reproductive status of female worms. A multiple sequence alignment showed 10 SNPs that were polymorphic and analysed for any associations. Three of these SNPs were statistically significantly associated with a poor response phenotype i.e. two at positions 1308C/T (P = 0.016) and 1545A/G (P = 0.008) within β-tub and one at position 5546A/G (P = 0.023) within P-gp. Within the β-tub, there was selection at position 1308C/T and some genotypes were present in good responders but absent in poor responders, vice versa. The heterozygosity was found to be reduced within worms sampled from Kintampo/Pru districts compared to those from Kpandai district. Although the biannual treatment in Ghana has made an impact, transmission still exists within some communities, and this is suggested to be driven by a few sub-optimally responding female worms in each community. These sub-optimal responses are also associated with some level of genetic changes. Regular monitoring of parasite responses to ivermectin treatment is necessary to avoid a completely resistant population emerging. Based on these findings, it is uncertain if increasing the frequency of ivermectin treatment (in Ghana) will be sufficient to meet the World Health Organization’s goals of onchocerciasis elimination by 2025.