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

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

doi:10.3390/pathogens10020188

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

¹,

⁴,

⁴,

⁵

⁶,

⁷

⁷

^1,2,†

and

Ulrike Loderstädt

^8,*,†

Department of Microbiology and Hospital Hygiene, Bundeswehr Hospital Hamburg, 20359 Hamburg, Germany

Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, 18057 Rostock, Germany

Laboratory for Medical Microbiology and Immunology, Elisabeth Tweesteden Hospital, 5042 AD Tilburg, The Netherlands

⁴

TIB MOLBIOL, 12103 Berlin, Germany

⁵

Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, 30559 Hannover, Germany

⁶

Medical Mission Institute, 97074 Würzburg, Germany

⁷

Infectious Disease Epidemiology Department, Bernhard Nocht Institute for Tropical Medicine Hamburg, 20359 Hamburg, Germany

⁸

Department of Hospital Hygiene & Infectious Diseases, University Medicine Göttingen, 37075 Göttingen, Germany

Author to whom correspondence should be addressed.

^†

Hagen Frickmann and Ulrike Loderstädt contributed equally to this work.

Academic Editors: Roberto Amerigo Papini and Hans-Peter Fuehrer

Pathogens 2021, 10(2), 188; https://doi.org/10.3390/pathogens10020188

Received: 8 January 2021 / Revised: 27 January 2021 / Accepted: 5 February 2021 / Published: 9 February 2021

(This article belongs to the Special Issue Zoonotic Parasitoses)

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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. View Full-Text

Keywords: helminths; stool samples; consensus; nematodes; trematodes; cestodes; qPCR; test comparison

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

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MDPI and ACS Style

Hoffmann, T.; Hahn, A.; Verweij, J.J.; Leboulle, G.; Landt, O.; Strube, C.; Kann, S.; Dekker, D.; May, J.; Frickmann, H.; Loderstädt, U. Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples. Pathogens 2021, 10, 188. https://doi.org/10.3390/pathogens10020188

AMA Style

Hoffmann T, Hahn A, Verweij JJ, Leboulle G, Landt O, Strube C, Kann S, Dekker D, May J, Frickmann H, Loderstädt U. Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples. Pathogens. 2021; 10(2):188. https://doi.org/10.3390/pathogens10020188

Chicago/Turabian Style

Hoffmann, Tanja, Andreas Hahn, Jaco J. Verweij, Gérard Leboulle, Olfert Landt, Christina Strube, Simone Kann, Denise Dekker, Jürgen May, Hagen Frickmann, and Ulrike Loderstädt. 2021. "Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples" Pathogens 10, no. 2: 188. https://doi.org/10.3390/pathogens10020188

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

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