Evaluation of a New Device for Simplifying and Standardizing Stool Sample Preparation for Viral Molecular Testing with Limited Hands-On Time

Abstract

Sensitive molecular assays have greatly improved the diagnosis of viral gastroenteritis. However, the proper preparation of stool samples for clinical testing remains an issue. bioMérieux has developed a stool preprocessing device (SPD) that includes a spoon for calibrated sampling and a vial containing buffer, glass beads, and two filters. The resulting stool filtrate is used for nucleic acid extraction. The purpose of this study was to evaluate the performance of the SPD for the quantification of human adenovirus (HAdV) DNA in stool samples collected from hematopoietic stem cell transplant (HSCT) recipients. HAdV DNA was quantified with the Adenovirus R-gene kit. The suitability of the device to reproducibly quantify HAdV DNA in stools using different extraction platforms (easyMAG and QIAsymphony) was determined using archived HAdV-positive stool samples. Coefficients of variation of HAdV DNA quantifications ranged from 1.79% to 1.83%, and no difference in quantification was observed between the two extraction systems. The HAdV DNA limit of quantification using the SPD was 3.75 log 10 copies/g of stool. HAdV DNA quantification using the SPD was then compared to that of the routine preprocessing technique on 75 fresh stool samples collected prospectively from pediatric HSCT recipients at risk for HAdV infections. Thirty-eight samples were HAdV DNA positive with both the SPD and routine preprocessing methods. HAdV DNA loads were on average 1.14-log 10 copies/g of stool higher with the SPD (P < 0.0001) than with routine methods. This new device enabled a standardized preparation of stool samples in <5 min and a reproducible and sensitive quantification of HAdV DNA. The use of the SPD for the detection of other gastrointestinal infections warrants further evaluation. PCR is a very rapid and reliable tool for molecular biologybased diagnosis of a variety of infectious diseases, using a wide range of clinical sample types (1).However, stool samples are among the most difficult clinical samples to process for molecular pathogen detection because of the presence of very potent inhibitors of nucleic acid (NA) amplification that are often coextracted along with pathogen NAs (2-4). Bile salts, hemoglobin, polysaccharides, heme, and bilirubin have been identified as factors that inhibit amplification assays (3, 5-8). Inhibitory effects can be reduced by adding amplification facilitators, such as bovine serum albumin, to the PCR mixture (9) by using thermostable polymerases that are more resistant to PCR inhibition (10), or by using more efficient processes for extracting NAs from stool samples (11). The efficiency of NA extraction and purification has been shown to influence the sensitivity, reproducibility, and accuracy of NA amplification-based target detection (12).Due to the potential presence of amplification inhibitors, sample preprocessing before NA extraction is an essential step to improve amplification-based pathogen detection from stool samples. Currently, stool preparation is not standardized and can be labor-...

Clinical and virological data of the first cases of COVID-19 in Europe: a case series

“…14 When a sample (respiratory samples, plasma, or stool) was positive with RdRp-IP1, quantification of the number of RNA copies was done according to a scale ranging from 10³ to 10⁶ copies per μL. The viral load in stools was calculated as previously described 15 and expressed in number of RNA copies per g of stool. The quality of nasopharyngeal swabs was checked using the CELL Control r-gene kit (bioMérieux).…”

“…This kit is provided with quantified plasmid for cellular quantification. All viral loads for respiratory samples were calculated with the same method as for stools 15 and normalised according to the cellular quantification as the number of RNA copies per 1000 cells. All positive plasma samples were quantified…”

A unique and reliable fecal DNA extraction method for 16S rRNA gene and shotgun metagenomic sequencing in the analysis of the human gut microbiome

“…06, Macherey-Nagel), the DNeasy PowerLyzer PowerSoil Kit (#12855-100, protocol 07272016, QIAGEN), the QIAamp Fast DNA Stool kit (#51604, QIAGEN, protocol modi ed from [36]) and the ZymoBIOMICS DNA Mini kit (#D4300, protocol 1.1.0, ZymoResearch). These protocols were also tested in combination with a stool preprocessing device (SPD, #421061, bioMérieux, [51]). This device was designed to facilitate and standardize fecal sample preparation before nucleic acid extraction.…”

“…Surprisingly, few studies have reported the use of commercial devices to standardize the handling of fecal samples prior to DNA extraction. [31,51,52]. Also, as the cell wall of Gram-positive bacteria is composed of a thick layer of peptidoglycan, bead-beating is now recommended to improve the lysis [53,54].…”

“…To address these considerations, our study evaluated four commercially available DNA extraction methods, using both SMS and 16S rRNA amplicon sequencing. These protocols were tested as recommended by the manufacturers, but also with an upstream stool preprocessing device (SPD), designed to facilitate DNA extraction [51]. The protocols were evaluated according to wet-lab as well as dry-lab criteria, using nine healthy individuals and nine Clostridium di cile infected patients.…”