Genotypic analysis of β-tubulin in Onchocerca volvulus from communities and individuals showing poor parasitological response to ivermectin treatment.

Ivermectin (IVM) has been in operational use for the control of onchocerciasis for two decades and remains the only drug of choice. To investigate the parasitological responses and genetic profile of Onchocerca volvulus, we carried out a 21 month epidemiological study to determine the response of the parasite to IVM in 10 Ghanaian endemic communities. Onchocerca nodules were surgically removed from patients in three IVM response categories (good, intermediate and poor) and one IVM naïve community. DNA from adult worms was analyzed to determine any association between genotype and IVM response phenotypic. Embryogramme analysis showed significantly higher reproductive activity in worms from poor response communities, which had up to 41% of females with live stretched microfilaria (mf) in utero, despite IVM treatment, compared with good response communities, which had no intra-uterine stretched mf. β-tubulin isotype 1 gene has been shown to be linked to IVM selection in O. volvulus and also known to be associated with IVM resistance in veterinary nematodes. We have genotyped the full length genomic DNA sequence of the β-tubulin gene from 127 adult worms obtained from the four community categories. We found SNPs at 24 sites over the entire 3696 bp. Eight of the SNPs occurred at significantly higher (p < 0.05) frequencies in the poor response communities compared with the good response communities and the IVM naïve community. Phenotypic and genotypic analyses show that IVM resistance has been selected and the genotype (1183GG/1188CC/1308TT/1545GG) was strongly associated with the resistance phenotype. Since the region in the β-tubulin gene where these four SNPs occur is within 362 bp, it is feasible to develop a genetic marker for the early detection of IVM resistance.