Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples.

Tanja Hoffmann,Simone Kann,Jaco J Verweij,Olfert Landt,Andreas Hahn,Gérard Leboulle,Hagen Frickmann,Ulrike Loderstädt,Denise Dekker,Christina Strube,Jürgen May

doi:10.3390/pathogens10020188

Abstract

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

Highlights

The global burden of helminth infections is still considerable [1,2,3,4,5,6,7,8,9,10,11], reliable microscopic diagnosis requires experience that is scarcely available in peripheral laboratories in settings of nonendemicity apart from reference centers
While superior sensitivity of real-time PCR for protozoan parasites compared to microscopy is considered to be well established [31], reliability of diagnostic real-time PCR for helminths is believed to depend on the harshness of nucleic acid extraction from strong-shelled eggs or cuticle cells [32,33,34]
When applied with helminth DNA, individual cross-reactions of the group specific helminth real-time PCRs were seen. Such cross-reactions were associated with comparably high cycle threshold (Ct) values

Summary

Introduction

The global burden of helminth infections is still considerable [1,2,3,4,5,6,7,8,9,10,11], reliable microscopic diagnosis requires experience that is scarcely available in peripheral laboratories in settings of nonendemicity apart from reference centers. Multiple genus- and species-specific PCRs and real-time PCRs [12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28] have been introduced for helminths. While superior sensitivity of real-time PCR for protozoan parasites compared to microscopy is considered to be well established [31], reliability of diagnostic real-time PCR for helminths is believed to depend on the harshness of nucleic acid extraction from strong-shelled eggs or cuticle cells [32,33,34]. Assessments of nucleic acid extraction schemes are usually performed only for specific helminth species, so it is difficult to draw general conclusions

Objectives

Methods

Results

Discussion

Conclusion