Harmonization of PCR-based detection of intestinal pathogens: experiences from the Dutch external quality assessment scheme on molecular diagnosis of protozoa in stool samples

Theo A. Schuurs; Rob Koelewijn; Eric A.T. Brienen; Titia Kortbeek; Theo G. Mank; Bert Mulder; Foekje F. Stelma; Lisette van Lieshout; Jaap J. van Hellemond

doi:10.1515/cclm-2017-1057

Article

Harmonization of PCR-based detection of intestinal pathogens: experiences from the Dutch external quality assessment scheme on molecular diagnosis of protozoa in stool samples

Theo A. Schuurs , Rob Koelewijn , Eric A.T. Brienen , Titia Kortbeek , Theo G. Mank , Bert Mulder , Foekje F. Stelma , Lisette van Lieshout and Jaap J. van Hellemond

Published/Copyright: February 16, 2018

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 56 Issue 10

Abstract

Background:

Real-time PCR methods are increasingly used in routine patient care settings not only to determine the presence or absence of pathogens in patient materials, but also to obtain semiquantitative results to estimate the pathogen load. However, it is so far unknown how well these methods are harmonized among different laboratories.

Methods:

Sets of stool samples were distributed three to four times per year to ca. 25–40 participating laboratories within the European Union as part of an external quality assessment scheme (EQAS) for the detection of gastrointestinal protozoa. This paper presents the results obtained over a 3-year period for Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, Cryptosporidium species and Dientamoeba fragilis.

Results:

Although both false-positive and false-negative results were reported, the overall sensitivity and specificity were high. The substantial differences in the quantitative output of the real-time PCR assays could be traced back to differences in DNA isolation procedures between different laboratories.

Conclusions:

Participation in an EQAS proved to be important as it provides information on how the real-time PCR methods used by the participant compares to the generally reported results and indicates how procedures could be improved. Semiquantitative results of real-time PCR methods are not exchangeable between laboratories as long as the diagnostic procedures are not harmonized. Intralaboratory comparison of semiquantitative real-time PCR results seems only possible by the use of calibration curves derived from well-validated standards in clinical material and not by spiking solutions with purified DNA.

Keywords: Cryptosporidium; Dientamoeba fragilis; Entamoeba histolytica; external quality assessment scheme; Giardia lamblia; intestinal protozoa; real-time PCR

Corresponding author: Dr. Jaap J. van Hellemond, Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Centre, PO Box 2040, 3000 CA Rotterdam, the Netherlands, Phone: +31 10 7033510, Fax: +31 10 7033875

aCurrent board members of the Parasitology Section of the Dutch Foundation for Quality Assessment in Medical Laboratories (SKML).

Acknowledgments

Dr. D.L. Borin is kindly acknowledged for critically reading the manuscript, and Mrs. N. van der Ham is thanked for excellent technical support in preparation of the EQAS samples.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Received: 2017-11-14

Accepted: 2018-01-23

Published Online: 2018-02-16

Published in Print: 2018-09-25

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Articles in the same Issue

https://doi.org/10.1515/cclm-2017-1057

Keywords for this article

Cryptosporidium; Dientamoeba fragilis; Entamoeba histolytica; external quality assessment scheme; Giardia lamblia; intestinal protozoa; real-time PCR