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

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.

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

“…It is not hard to imagine that the latter is especially important in the isolation of parasite DNA from a complex matrix such as feces (11). Heating of the stool specimen and the addition of absorbent substances such as polyvinyl polypyrrolidone during the DNA isolation procedure or the addition of inhibition factorbinding substances, such as bovine serum albumin (BSA), or inhibitor-resistant DNA polymerases in the PCR mixture can be used to prevent inhibition of the amplification reaction (12)(13)(14)(15).…”

“…With regard to the first aspect, it appears that the efficient release of nucleic acids depends on a balanced combination of actions in the DNA isolation procedure. For example, some papers mention specifically that additional mechanical disruption is needed for the efficient isolation of DNA from Trichuris eggs or Entamoeba cysts, while others use a standard isolation protocol without mention of additional rigorous steps to break down the egg shell or cyst wall (15,(17)(18)(19). Another example is the negative effect of preserving feces in formalin or sodium acetate-acetic acid-formalin (SAF) on the specific amplification of Entamoeba, Giardia, and Cryptosporidium DNAs; this effect increases with the duration of fixation (20)(21)(22)(23).…”

Isothermal Diagnostic Assays for Monitoring Single Nucleotide Polymorphisms in Necator americanus Associated with Benzimidazole Drug Resistance

“…To improve the performance of SmartAmp2 assay in fecal samples, sample collection and processing should be simple and ensure high DNA yields. Preliminary enrichment of eggs through sugar flotation followed by repeated cycles of freeze/boiling to crack the egg shell and liberate DNA [43] may significantly improve DNA recovery, assay sensitivity and reproducibility. Commercial DNA extraction methods are reliable and efficient for removing PCR inhibitors present in fecal samples; however, these methods use a small amount of feces and this could affect the sensitivity of the detection assay.…”

Multiplex real-time PCR monitoring of intestinal helminths in humans reveals widespread polyparasitism in Northern Samar, the Philippines

“…However, this species was not included in the initial multiplex assay undertaken here as the egg shells of T. trichiura are known to be resistant to standard copro-extraction methods (Areekul et al, 2010;Demeler et al, 2013;Mejia et al, 2013). As such, these eggs require an additional homogenisation step prior to extraction to break open the eggs and effectively extract the DNA (Demeler et al, 2013). The human faecal samples used in this study were originally used to extract DNA for qPCR studies on S. japonicum (Gordon et al, 2015a,b) so the additional step required to extract DNA for T. trichiura eggs was not performed.…”