Using deep amplicon sequencing as a molecular xenomonitoring approach for detecting filarial nematodes in biting arthropod vectors

Filarial nematodes are an important group of parasites that impact public, veterinary, and wildlife health globally. In order to understand these impacts and minimize their effects, scientists use molecular xenomonitoring techniques to understand their distribution and track elimination efforts. However, these molecular techniques can have narrow diagnostic capacity due to their species-specific approach which limits our understanding of important co-endemic filarial nematodes. Next-generation sequencing offers the ability to detect multiple species of coinfecting filarial nematodes and thus improve are ability to monitor, treat, and eliminate these pathogens. In this paper, we have developed a deep amplicon sequencing approach using filarial nematode primers targeting the cytochrome oxidase c subunit 1 (coxI) gene. To replicate molecular xenomonitoring conditions, third stage larvae (L3) of three species of filarioid nematodes (Brugia malayi, Brugia pahangi, Dirofilaria immitis) were spiked in different proportions to pools comprising various amounts of female Aedes aegypti mosquitoes (0, 10, 50, 100). Each pool was subjected to DNA extraction and Oxford Nanopore Technologies (ONT) deep amplicon sequencing protocols. Two sets of demultiplexing pipelines were utilized to optimize this novel approach, each reaching, 92.71% and 97.92% accuracy in identification of species composition across mock pools. However, in heterogenous pools, filarial species D. immitis exhibited an overrepresentation of reads and B. pahangi an underrepresentation of reads. We discuss reasons for recount biases and how this new molecular xenomonitoring tool could be implemented to serve public health, veterinary medicine, and scientific advancement.