Improved NAAT assay for the diagnosis of onchocerciasis and its use for detection of circulating cell free DNA

Abstract

AbstractMass drug administration (MDA) programs aimed at control and elimination of onchocerciasis relies on annual or semi-annual distribution of Ivermectin (IVM) that target the microfialarial (mf) stage of the parasiteOnchocerca volvulus. The co-endemicity of onchocerciasis with other filarial species often leads to interruptions in the control programs. A much-needed tool for the elimination efforts are sensitive diagnostic assays that can help differentiate from other co-endemic filarial infections or xenomonitoring approaches. qPCR assays targeting highly repeated elements in the genomes have allowed for increased sensitivity of detection that are pathogen-specific. Utilizing NGS data, qPCR assays were designed to target 15 highly repeated targets fromO. volvulusand 11 fromO. ochengi. The two most sensitive and specific repeats Ov15R and Ov16R fromO. volvulusand OoR1 and OoR5 fromO. ochengi, were selected for further testing.The analytical sensitivity of both Ov15R and Ov16R were similar with limits of detection (LOD) at 1 fg ofO. volvulusgenomic DNA (gDNA), and 100% specificity and ∼32-fold sensitive than O-150 qPCR. Similarly, OoR1 and OoR5 had LOD of 100 fg ofO. ochengigDNA. Using DNA obtained previously from skin snips, Ov16R identified 17 additional samples as positive for infections when compared to O-150.Further, to eliminate the need for time-dependent sample acquisition, we evaluated the efficiency of plasma-derived circulating cell-free DNA (ccfDNA) to detectO. volvulusinfections and as a potential modality for testing cure of infection. Plasma-derived ccfDNA failed to amplify Ov16R. In contrast using a small set of urine-derived ccfDNA samples from Ov-infected individuals had varying levels of detectability as early as 12-24 hours post-treatment. To enable processing of larger volumes of urine for better sensitivity, a modified chitosan-based filter technique was developed as proof-of-concept that efficiently captured gDNA from 1-15ml of urine.Interestingly, Ov15R, Ov16R and O-150 map to the same contigs ofO. volvulusgenome. This prompted the redesign of O-150 qPCR that resulted in O-150-New qPCR assay that performs on par with Ov15R/Ov16R, thus offering more sensitive and specific diagnosis ofO. volvulusthat can easily be configured to field-friendly formats such as LAMP or RPA.