Improved Nucleic Acid Amplification Test for the Diagnosis of Onchocerciasis and Its Use for Detection of Circulating Cell-free DNA

Background The coendemicity of onchocerciasis with other filariae warrants a better diagnostic tool for elimination efforts that are highly sensitive and specific for use in surveillance and xenomonitoring.Methods Based on next-generation sequencing data, quantitative polymerase chain reaction (qPCR) assays were designed for 15 highly repeated targets from Onchocerca volvulus (Ov) and 11 from Onchocerca ochengi. The 2 most promising repeats Ov15R and Ov16R from Ov and OoR1 and OoR5 from O. ochengi, were selected for further testing.Results The analytic sensitivity of Ov15R and Ov16R was similar, with limits of detection at 1 fg and specificity approaching 100%. Using DNA obtained previously from skin snips of participants infected with Ov, Ov16R identified 17 additional samples as positive for Ov infections when compared with the gold standard O-150. Although Ov16R failed to detect circulating cell-free DNA (ccfDNA) in the plasma of individuals infected with Ov, 1-mL urine samples were variably positive for ccfDNA. Interestingly, plasma levels of ccfDNA were shown to be easily measurable as early as 12 to 24 hours following treatment. To enable processing of larger volumes of urine for better sensitivity, a chitosan-based filter technique was developed that efficiently captured ccfDNA from 1 to 15 mL of urine. Interestingly, Ov15R, Ov16R, and O-150 map to the same region(s) of the Ov genome, prompting a redesign of the standard O-150 qPCR. This resulted in a new O-150 assay that performs on par with Ov15R/Ov16R.Conclusions Each of these assays dramatically improve detection of Ov DNA and can easily be configured to field-friendly isothermal formats. Novel repeat-based qPCR targets offer a highly sensitive diagnosis of onchocerciasis that can help not only diagnosis infection but also likely monitor the cure for patients undergoing antifilarial treatment.