Additional Egg Disruption Techniques Facilitate Better DNA Yield for Species Level Identification of Ascaris lumbricoides and Trichuris trichiura

Background and Objective: Major species in soil transmitted helminths (STHs) infections are roundworms (Ascaris lumbricoides), whipworms (Trichuris trichiura) and hookworms (Necator americanus and Ancylostoma duodenale). Detection of these STHs is routinely done by microscopic examination from stool samples as it relatively simple and less expensive, however species level identification needs nucleic acid-based diagnostic techniques which can be challenging. Hence, this study focused on employing physical and chemical methods for the disruption of the roundworm and whipworm eggshells prior to DNA extraction. Materials and Methods: Primarily solid particles from stool were removed by passing through two layered damped gauze. Next, the eggs in the filtrate were concentrated by flotation in salt/sugar solutions. The eggs were counted and subjected to different disruption procedures like freeze thaw cycles, sonication, boiling and microwaving, more than one method can be used for eggs disruption. The disrupted eggs were subjected to DNA extraction using commercial kits following manufacturers protocol. Results: The additional disruptions methods yielded good quality and quantity of DNA. Among the methods used, incubation of eggs in high density solutions overnight showed higher DNA yield for Trichuris trichiura. Boiling the eggs for a brief time followed by freeze thaw cycles depicted higher concentration of quality DNA for Ascaris lumbricoides. Due to the nature of the eggshells from different species-specific methods needs to be standardized to get better DNA yield.