Multicenter Comparative Study of Six Cryptosporidium parvum DNA Extraction Protocols Including Mechanical Pretreatment from Stool Samples.

Nicolas Valeix,Louise Basmaciyan,Stéphane Valot,Céline Nourrisson,Loic Favennec,Frederic Dalle,Damien Costa,Romy Razakandrainibe,Anne Vincent,Philippe Poirier,Florence Robert-Gangneux,Bruno Pereira,Emilie Fréalle

doi:10.3390/microorganisms8091450

Abstract

Background: Nowadays, many commercial kits allow the detection of Cryptosporidium sp. in stool samples after deoxyribonucleic acid (DNA) extraction. Protocols of stool pretreatment have been proposed to optimize oocysts’ DNA extraction. Among them, mechanical grinding was reported to improve the performance of Cryptosporidium oocysts’ DNA extraction. Methods: A multicenter comparative study was conducted within the framework of the French National Reference Center-Expert Laboratory for Cryptosporidiosis. Six extraction systems (i.e., manual or automated) associated with various mechanical pretreatment protocols, were compared for the Cryptosporidium parvum oocyst’ DNA extraction, before amplification using the same real-time PCR method targeting. Results: The sensitivity of real-time PCR assay was unequally impacted by the pretreatment/extraction protocol. We observed significant differences for the lowest concentrations of C. parvum oocysts (i.e., 0–94.4% and 33.3–100% respectively for 10 and 50 oocysts/mL). All in all, the protocol using Quick DNA Fecal/Soil Microbe-Miniprep® manual kit showed the best performances. In addition, optimal performances of mechanical pretreatment were obtained by combining a grinding duration of 60 s with a speed of 4 m/s using Fastprep24® with Lysing Matrix E®. Conclusions: Sample pretreatment, as well as the extraction method, needs to be properly adapted to improve the diagnostic performances of the C. parvum DNA amplification methods.

Highlights

The protozoan parasite Cryptosporidium sp. infects a wide range of vertebrate hosts, including humans, in whom it behaves as an opportunistic pathogen
The performances in C. parvum deoxyribonucleic acid (DNA) amplification were variable depending on the protocol used
We further investigated the influence of the speed and duration of the grinding step on the extraction performances of one of the methods investigated in the present work, namely the easyMAG® system (EM) system that combines the grinder Fastprep 24 (MP BiomedicalsTM) with Lysing Matrix E (MP BiomedicalsTM)

Summary

Introduction

The protozoan parasite Cryptosporidium sp. infects a wide range of vertebrate hosts, including humans, in whom it behaves as an opportunistic pathogen. Cryptosporidiosis is a current public health concern, with waterborne and foodborne outbreaks occurring each year worldwide [4]. In this context, efficient biological methods for cryptosporidiosis diagnosis are needed. Protocols of stool pretreatment have been proposed to optimize oocysts’ DNA extraction. Mechanical grinding was reported to improve the performance of Cryptosporidium oocysts’ DNA extraction. Six extraction systems (i.e., manual or automated) associated with various mechanical pretreatment protocols, were compared for the Cryptosporidium parvum oocyst’ DNA extraction, before amplification using the same real-time PCR method targeting. Conclusions: Sample pretreatment, as well as the extraction method, needs to be properly adapted to improve the diagnostic performances of the C. parvum DNA amplification methods

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Conclusion