Discrimination of Gastrointestinal Nematode Eggs from Crude Fecal Egg Preparations by Inhibitor-Resistant Conventional and Real-Time PCR

Janina Demeler,Georg Von Samson-Himmelstjerna,Giuseppe Cringoli,Jean Bosco Gahutu,Jürgen Krücken,Davide Ianiello,Sonja Wolken,Sabrina Ramünke,Laura Rinaldi,Clotilde K S Carlow

doi:10.1371/journal.pone.0061285

Abstract

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles – only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM.

Highlights

Gastrointestinal nematodes are important pathogens in veterinary and tropical medicine and due to the zoonotic potential of some species (e.g. Toxocara spp., Ascaris suum, Ancylostoma caninum), close collaboration between veterinarians and tropical disease specialists would improve intervention measures
The direct PCR method developed in this project has the potential to allow highly sensitive molecular diagnosis of gastrointestinal nematodes with importance for veterinary and human tropical medicine at decreased costs
The results presented here show that PCR products produced by d-PCR are amenable to analysis by restriction fragment length polymorphism (RFLP), Sanger sequencing and real-time PCR with fluorescent dyes binding double-stranded DNA followed by high resolution melt (HRM) analysis to rapidly identify particular pathogens

Summary

Introduction

Gastrointestinal nematodes are important pathogens in veterinary and tropical medicine and due to the zoonotic potential of some species (e.g. Toxocara spp., Ascaris suum, Ancylostoma caninum), close collaboration between veterinarians and tropical disease specialists would improve intervention measures. Traditional coproscopic diagnostic tools offer the advantage of being rapid and inexpensive, allowing either sensitive qualitative (e.g. simple flotation in tube, ether-concentration methods) or moderately sensitive quantitative (e.g. McMaster and Kato-Katz methods) determination of the infection status of humans and animals including e.g. ruminants, equines, cats and dogs. Due to the fact that many nematode eggs are virtually indistinguishable using conventional microscopy, there is an increasing need to develop new diagnostic tools based on genetic discrimination of these organisms [5,6,7]. This is true for the order Strongylida that includes many species frequently found to co-parasitize the same host. Different strongylid parasite species differ substantially in their pathogenicity and require different interpretation of fecal egg counts before treatment decisions and molecular techniques are required to identify the species present

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Discussion

Conclusion