Evaluation of different stool extraction methods for metabolomics measurements in human fecal samples

AbstractBackgroundMeasurement of metabolomics in human stool samples is of great interest for a broad range of applications in biomedical research including early detection of colorectal neoplasms. However, due to the complexity of metabolites there is no consensus on how to process samples for stool metabolomics measurements to obtain a broad coverage of hydrophilic and hydrophobic substances.MethodsWe used frozen stool samples (50mg) from healthy study participants. Stool samples were processed after thawing using 8 different processing protocols and different solvents. Metabolites were measured afterwards using the MxP® Quant 500 kit (Biocrates). The best performing protocol was subsequently applied to compare stool samples of participants with different dietary habits.ResultsIn this study, we were able to determine up to 340 metabolites of various chemical classes extracted from stool samples of healthy study participants with 8 different protocols. Polar metabolites such as amino acids could be measured with each method while other metabolite classes, particular lipid species, are more dependent on the solvent or combination of solvents used. Only a small number of triglycerides or acylcarnitines were detected in human feces. Extraction efficiency was higher for protocols using isopropanol or those using ethanol or methanol and MTBE including an evaporation and concentration step than for other protocols. We detected significant fecal metabolite differences between vegetarians, semi-vegetarians and non-vegetarians.ConclusionFor the evaluation of metabolites in fecal samples we found protocols using solvents like isopropanol and those using ethanol or methanol and MTBE including an evaporation and concentration step to be superior over others tested in this study.

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Discovery of novel astrovirus genotype species in small ruminants

PeerJ ◽

10.7717/peerj.7338 ◽

2019 ◽

Vol 7 ◽

pp. e7338

Author(s):

Ronja V. Kauer ◽

Michel C. Koch ◽

Melanie M. Hierweger ◽

Simea Werder ◽

Céline L. Boujon ◽

...

Keyword(s):

Phylogenetic Analyses ◽

Rna Extraction ◽

Small Ruminants ◽

Interspecies Transmission ◽

Fecal Samples ◽

Fecal Shedding ◽

Ruminant Species ◽

Stool Samples ◽

Full Length Genome ◽

Human Stool

Astroviruses (AstV) are single-stranded, positive-sense RNA viruses, best known for causing diarrhea in humans and are also found in many other mammals; in those, the relevance in gastroenteritis remains unclear. Recently described neurotropic AstV showed associations with encephalitis in humans as well as in other mammals. In Switzerland, two different neurotropic AstV were identified in cattle, as well as one in a sheep. The high genetic similarity between the ovine and one of the bovine AstV strengthens the hypothesis of an interspecies transmission. In humans, AstV associated with encephalitis were found also in human stool samples, suggesting that in these patients the infection spreads from the gastrointestinal tract to the brain under certain conditions, such as immunosuppression. Whether a similar pathogenesis occurs in ruminants remains unknown. The aims of this study were (1) the investigation of the potential occurrence of neurotropic AstV in feces samples, (2) the discovery and analysis of so far unknown AstV in small ruminants and other ruminant species’ fecal samples and (3) the examination of a potential interspecies transmission of AstV. To achieve these aims, RNA extraction out of 164 fecal samples from different ruminant species was performed and all samples were screened for known neurotropic AstV occurring in Switzerland, as well as for various AstV using RT-PCR. Positive tested samples were submitted to next generation sequencing. The generated sequences were compared to nucleotide- and amino acid databases, virus properties were identified, and phylogenetic analyses as well as recombination analysis were performed. The excretion of neurotropic AstV in small ruminants’ feces could not be demonstrated, but this work suggests the first identification of AstV in goats as well as the discovery of multiple and highly diverse new genetic variants in small ruminants, which lead to a classification into novel genotype-species. Additionally, the prediction of multiple recombination events in four of five newly discovered full or almost full-length genome sequences suggests a plausible interspecies transmission. The findings point out the occurrence and fecal shedding of previously unknown AstV in sheep and goats and pave the way towards a better understanding of the diversity and transmission of AstV in small ruminants.

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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

Pathogens ◽

10.3390/pathogens10020188 ◽

2021 ◽

Vol 10 (2) ◽

pp. 188

Author(s):

Tanja Hoffmann ◽

Andreas Hahn ◽

Jaco J. Verweij ◽

Gérard Leboulle ◽

Olfert Landt ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Bead Beating ◽

Stool Samples ◽

Pcr Assays ◽

Human Stool

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.

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Detection of SARS-CoV-2 from patient fecal samples by whole genome sequencing

Gut Pathogens ◽

10.1186/s13099-021-00398-5 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Andreas Papoutsis ◽

Thomas Borody ◽

Siba Dolai ◽

Jordan Daniels ◽

Skylar Steinberg ◽

...

Keyword(s):

Next Generation Sequencing ◽

Nasopharyngeal Swab ◽

Whole Genome ◽

Rt Pcr ◽

Whole Genome Analysis ◽

Fecal Samples ◽

Oral Transmission ◽

Study Participants ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment next-generation sequencing (NGS) from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal–oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication. Trial registration ClinicalTrials.gov, NCT04359836, Registered 24 April 2020, https://clinicaltrials.gov/ct2/show/NCT04359836?term=NCT04359836&draw=2&rank=1).

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Multicenter Evaluation of an ELISA for the Detection of Cryptosporidium spp. Antigen in Clinical Human Stool Samples

Microorganisms ◽

10.3390/microorganisms9020209 ◽

2021 ◽

Vol 9 (2) ◽

pp. 209

Author(s):

Romy Razakandrainibe ◽

Célia Mérat ◽

Nathalie Kapel ◽

Marc Sautour ◽

Karine Guyot ◽

...

Keyword(s):

Large Scale ◽

Cryptosporidium Oocyst ◽

Clinical Importance ◽

Epidemiological Studies ◽

Elisa Test ◽

Enzyme Linked Immunosorbent Assay ◽

Cryptosporidium Spp ◽

Stool Samples ◽

Conventional Microscopy ◽

Human Stool

Human cryptosporidiosis remains underdiagnosed, and rapid/accurate diagnosis is of clinical importance. Diagnosis of the Cryptosporidium oocyst in stool samples by conventional microscopy is labor-intensive, time-consuming, and requires skillful experience. Thus, we aimed to evaluate the usefulness of a coproantigen enzyme-linked immunosorbent assay (ELISA) test in detecting Cryptosporidium spp. from fecal specimens. For this aim, we evaluated the performances of a commercial ELISA (CoproELISA Cryptosporidium kit, Savyon Diagnostics, Israel) for the detection of Cryptosporidium spp. in random clinical stool samples through a multicenter study. The sensitivity and specificity for coproantigen ELISA were 98.86% and 94.32%, respectively. The coproantigen ELISA results indicate that the simple, rapid, reliable, and standardized immunoassay test is sensitive and specific for routine diagnosis, and may be useful for large-scale epidemiological studies of cryptosporidiosis.

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