Development of a Confirmatory PCR Assay to Detect Onchocerca volvulus in Pools of Vector Black Flies

Onchocerciasis, or river blindness, has historically represented one of the significant neglected tropical diseases on the planet in terms of socio-economic impact. The discovery that ivermectin was a safe and effective treatment for onchocerciasis, together with the decision of the manufacturer to donate the drug for the treatment of this disease became the basis for several large international programs to control and eventually eliminate the infection. These programs have managed to virtually eliminate transmission of the parasite causing Onchocerca volvulus from many foci in Africa and the Americas. Verifying that transmission has been halted requires sensitive and specific assays to detect the presence of the parasite. The gold standard to accomplish this has been to employ a PCR assay targeting a specific repeated sequence family encoded in the genome of O. volvulus to screen for the presence of the parasite in pools of vector black flies. While this assay is highly sensitive, obtaining the high specificity required to document an absence of transmission requires an independent confirmatory assay. To meet this need, an independent PCR assay targeting the cytochrome B (cytB) gene of the O. volvulus mitochondrion was developed. This assay could detect O. volvulus mitochondrial DNA purified by magnetic bead capture using the primers for the cytB gene and from the nuclear encoded repeated sequence DNA targeted in the primary assay. These preliminary data suggest that the mitochondrial PCR assay may be employed as a confirmatory assay to detect O. volvulus in pools of vector flies