scholarly journals Discovery of novel astrovirus genotype species in small ruminants

PeerJ ◽  
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.

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

2020 ◽  
Author(s):  
Vanessa Erben ◽  
Gernot Poschet ◽  
Petra Schrotz-King ◽  
Hermann Brenner
Keyword(s):  
Dietary Habits ◽  
Extraction Methods ◽  
Fecal Samples ◽  
Lipid Species ◽  
Hydrophobic Substances ◽  
Polar Metabolites ◽  
Concentration Step ◽  
Stool Samples ◽  
Study Participants ◽  
Human Stool

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.

scholarly journals Partial Genome Sequences of Human Norovirus Strains from Northeast Brazil

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Klarissa Miranda Guarines ◽  
Renata Pêssoa Germano Mendes ◽  
Jurandy Júnior Ferraz de Magalhães ◽  
Lindomar Pena
Keyword(s):  
Phylogenetic Analyses ◽  
Genome Sequences ◽  
Junction Region ◽  
Human Norovirus ◽  
Reading Frame ◽  
Stool Samples ◽  
Norovirus Gii ◽  
Partial Genome ◽  
Human Stool ◽  
Open Reading Frame 1

Noroviruses are the leading cause of human gastroenteritis worldwide. Here, we sequenced the open reading frame 1 (ORF1)-ORF2 junction region of norovirus strains isolated from 20 human stool samples. Samples were collected between 2014 and 2017 in Pernambuco State, Brazil. Phylogenetic analyses identified four norovirus GII genotypes circulating in this area of the country.

scholarly journals Validation and testing of a method for detection of SARS-CoV-2 RNA in healthy human stool

2020 ◽  
Author(s):  
Michael P. Coryell ◽  
Mikhail Iakiviak ◽  
Nicole Pereira ◽  
Pallavi P. Murugkar ◽  
Jason Rippe ◽  
...  
Keyword(s):  
Ambient Temperature ◽  
The United States ◽  
Fecal Microbiota ◽  
Viral Rna ◽  
Fecal Shedding ◽  
Donor Screening ◽  
Stool Samples ◽  
Monitoring Protocols ◽  
Development And Validation ◽  
Human Stool

Summary (Abstract)BackgroundFecal shedding of SARS-CoV-2 has raised concerns about transmission through fecal microbiota transplantation (FMT) procedures. While many tests have been authorized for diagnosis of COVID-19 using respiratory samples, no fully validated stool tests for detection of SARS-CoV-2 are currently available. We sought to adapt and validate an available test specifically for detection of SARS-CoV-2 in human stool.MethodsStool samples were spiked with inactivated SAR-CoV-2 virus for development and validation of the assay. A modified version of the CDC rRT-PCR SARS-CoV-2 test was used for detection of virus. Analytical sensitivity, assay reproducibility, and sample stability under a variety of storage conditions were assessed. We also performed the assay on stool samples collected from known COVID positive individuals.FindingsThe lower limit of detection (LoD) of the assay was found to be 3000 viral RNA copies per gram of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Samples were relatively stable in all buffers tested at both 4°C and ambient temperature, with the exception of storage in STAR buffer at ambient temperature. Assay sensitivity was slightly diminished in low-copy-number samples after a single freeze-thaw cycle at −80°C. Thirty contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, we detected SARS-CoV-2 RNA in the stool of known COVID-19 positive individuals using this method.InterpretationThis is a sensitive, reproducible, and validated assay for detection of SARS-CoV-2 RNA in human stool with potential uses in FMT donor screening, sewage monitoring, and further research into the impact of fecal shedding on the epidemiology of this pandemic.FundingNational Institute for Allergy and Infectious Diseases, NIH. Center for Biologics Evaluation and Research, FDA.Research in ContextEvidence before this studySince the onset of the COVID-19 pandemic, multiple studies have documented shedding of SARS-CoV-2 RNA in feces and considered the potential for fecal-oral transmission of this virus. This potential risk led to the U.S. Food and Drug Administration issuing a safety alert that contained the recommendation that no stool donated after December 1, 2019 be used for manufacture of Fecal Microbiota for Transplantation (FMT) products in the United States until such a time as sufficient screening procedures could be put in place to mitigate this risk.Added value of this studyHere, we report the development and validation of an assay specifically meant for the detection of SARS-CoV-2 RNA in the stool of healthy individuals. While studies have reported detection of viral RNA in stool previously, this is the first publication of a validated assay designed for this purpose.Implications of all the available evidenceThe work presented here provides a validated SARS-CoV-2 stool assay with potential application to FMT donor screening protocols, sewage monitoring protocols, as well as research studies assessing the role of stool shedding and transmission on the epidemiology of COVID-19.

The Frequency of HIV-1 Infection in Iranian Children and Determination of the Transmitted Drug Resistance in Treatment-Naïve Children

2020 ◽  
Vol 17 (6) ◽  
pp. 397-407
Author(s):  
Maryam Jarchi ◽  
Farah Bokharaei-Salim ◽  
Maryam Esghaei ◽  
Seyed Jalal Kiani ◽  
Fatemeh Jahanbakhsh ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Pediatric Patients ◽  
Phylogenetic Analyses ◽  
Rna Extraction ◽  
Transmitted Drug Resistance ◽  
The Arts ◽  
Treatment Naïve ◽  
Iranian Children ◽  
Hiv 1

Background: The advent of resistance-associated mutations in HIV-1 is a barrier to the success of the ARTs. Objective: In this study, the abundance of HIV-1 infection in Iranian children, and also detection of the TDR in naïve HIV-1 infected pediatric (under 12 years old) were evaluated. Materials: From June 2014 to January 2019, a total of 544 consecutive treatment-naïve HIV-1- infected individuals enrolled in this study. After RNA extraction, amplification, and sequencing of the HIV-1 pol gene, the DRM and phylogenetic analysis were successfully performed on the plasma specimens of the ART-naïve HIV-1-infected-children under 12 years old. The DRMs were recognized using the Stanford HIV Drug Resistance Database. Results: Out of the 544 evaluated treatment-naïve HIV-1-infected individuals, 15 (2.8%) cases were children under 12 years old. The phylogenetic analyses of the amplified region of pol gene indicated that all of the 15 HIV-1-infected pediatric patients were infected by CRF35_AD, and a total of 13.3% (2/15) of these children were infected with HIV-1 variants with SDRMs (one child harbored two related SDRMs [D67N, V179F], and another child had three related SDRMs [M184V, T215F, and K103N]), according to the last algorithm of the WHO. No PIs-related SDRMs were observed in HIV-1-infected children. Conclusion: The current study demonstrated that a total of 13.3% of treatment-naïve HIV-1-infected Iranian pediatrics (under 12 years old) were infected with HIV-1 variants with SDRMs. Therefore, it seems that screening to recognize resistance-associated mutations before the initiation of ARTs among Iranian children is essential for favorable medication efficacy and dependable prognosis.

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

Pathogens ◽  
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.

scholarly journals Global Distribution and Genetic Heterogeneity of Border Disease Virus

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 950
Author(s):  
Cecilia Righi ◽  
Stefano Petrini ◽  
Ilaria Pierini ◽  
Monica Giammarioli ◽  
Gian Mario De Mia
Keyword(s):  
Disease Virus ◽  
Significant Risk ◽  
Small Ruminants ◽  
Control Measures ◽  
Interspecies Transmission ◽  
Economic Losses ◽  
Border Disease Virus ◽  
Diarrhea Virus ◽  
Geographical Regions ◽  
Border Disease

Border disease virus (BDV) belongs to the genus Pestivirus of the family Flaviviridae. Interspecies transmission of BDV between sheep, cattle, and pigs occurs regularly, sometimes making diagnosis a challenge. BDV can yield substantial economic losses, including prenatal and postnatal infections in lambs, which are the primary source of infection and maintenance of the virus in the population. Since BDV is antigenically and genetically related to bovine viral diarrhea virus (BVDV), it might pose a significant risk to cattle, influencing BVDV eradication campaigns. Similarly, the presence of BDV in swine herds due to pestivirus spillover between small ruminants and pigs might cause uncertainty in classical swine fever virus (CSFV) diagnostics. Therefore, knowledge of BDV epidemiology in different geographical regions will help prevent its spread and optimize control measures. Previous epidemiological studies have shown that various BDV genotypes are predominant in different countries. This review provides an overview of the spread of BDV world-wide in different host species.

scholarly journals Genetic Diversity of Enteric Viruses in Children under Five Years Old in Gabon

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 545
Author(s):  
Gédéon Prince Manouana ◽  
Paul Alvyn Nguema-Moure ◽  
Mirabeau Mbong Ngwese ◽  
C.-Thomas Bock ◽  
Peter G. Kremsner ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Preventive Measures ◽  
Phylogenetic Analyses ◽  
Enteric Viruses ◽  
Study Cohort ◽  
Viral Agent ◽  
A Genome ◽  
Stool Samples ◽  
Pcr Techniques ◽  
High Diversity

Enteric viruses are the leading cause of diarrhea in children globally. Identifying viral agents and understanding their genetic diversity could help to develop effective preventive measures. This study aimed to determine the detection rate and genetic diversity of four enteric viruses in Gabonese children aged below five years. Stool samples from children <5 years with (n = 177) and without (n = 67) diarrhea were collected from April 2018 to November 2019. Norovirus, astrovirus, sapovirus, and aichivirus A were identified using PCR techniques followed by sequencing and phylogenetic analyses. At least one viral agent was identified in 23.2% and 14.9% of the symptomatic and asymptomatic participants, respectively. Norovirus (14.7%) and astrovirus (7.3%) were the most prevalent in children with diarrhea, whereas in the healthy group norovirus (9%) followed by the first reported aichivirus A in Gabon (6%) were predominant. The predominant norovirus genogroup was GII, consisting mostly of genotype GII.P31-GII.4 Sydney. Phylogenetic analysis of the 3CD region of the aichivirus A genome revealed the presence of two genotypes (A and C) in the study cohort. Astrovirus and sapovirus showed a high diversity, with five different astrovirus genotypes and four sapovirus genotypes, respectively. Our findings give new insights into the circulation and genetic diversity of enteric viruses in Gabonese children.

scholarly journals Multiple Types of Novel Enteric Bopiviruses (Picornaviridae) with the Possibility of Interspecies Transmission Identified from Cloven-Hoofed Domestic Livestock (Ovine, Caprine and Bovine) in Hungary

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 66
Author(s):  
Zoltán László ◽  
Péter Pankovics ◽  
Gábor Reuter ◽  
Attila Cságola ◽  
Ádám Bálint ◽  
...  
Keyword(s):  
Novel Species ◽  
Phylogenetic Analyses ◽  
Interspecies Transmission ◽  
Background Information ◽  
Type Ii ◽  
Rt Pcr ◽  
Faecal Samples ◽  
The Family ◽  
Domestic Livestock

Most picornaviruses of the family Picornaviridae are relatively well known, but there are certain “neglected” genera like Bopivirus, containing a single uncharacterised sequence (bopivirus A1, KM589358) with very limited background information. In this study, three novel picornaviruses provisionally called ovipi-, gopi- and bopivirus/Hun (MW298057-MW298059) from enteric samples of asymptomatic ovine, caprine and bovine respectively, were determined using RT-PCR and dye-terminator sequencing techniques. These monophyletic viruses share the same type II-like IRES, NPGP-type 2A, similar genome layout (4-3-4) and cre-localisations. Culture attempts of the study viruses, using six different cell lines, yielded no evidence of viral growth in vitro. Genomic and phylogenetic analyses show that bopivirus/Hun of bovine belongs to the species Bopivirus A, while the closely related ovine-origin ovipi- and caprine-origin gopivirus could belong to a novel species “Bopivirus B” in the genus Bopivirus. Epidemiological investigation of N = 269 faecal samples of livestock (ovine, caprine, bovine, swine and rabbit) from different farms in Hungary showed that bopiviruses were most prevalent among <12-month-old ovine, caprine and bovine, but undetectable in swine and rabbit. VP1 capsid-based phylogenetic analyses revealed the presence of multiple lineages/genotypes, including closely related ovine/caprine strains, suggesting the possibility of ovine–caprine interspecies transmission of certain bopiviruses.

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

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.

scholarly journals Prevalence and Genetic Characterisation of Human Sapovirus from Children with Diarrhoea in the Rural Areas of Vhembe District, South Africa, 2017–2020

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 393
Author(s):  
Mpho Magwalivha ◽  
Jean-Pierre Kabue Ngandu ◽  
Afsatou Ndama Traore ◽  
Natasha Potgieter
Keyword(s):  
South Africa ◽  
Rural Communities ◽  
Rural Areas ◽  
Rna Extraction ◽  
Diarrhoeal Disease ◽  
Positive Sample ◽  
Prevalent Strain ◽  
Stool Samples ◽  
Vhembe District ◽  
Pcr Targeting

Diarrhoeal disease is considered an important cause of morbidity and mortality in developing areas, and a large contributor to the burden of disease in children younger than five years of age. This study investigated the prevalence and genogroups of human sapovirus (SV) in children ≤5 years of age in rural communities of Vhembe district, South Africa. Between 2017 and 2020, a total of 284 stool samples were collected from children suffering with diarrhoea (n = 228) and from children without diarrhoea (n = 56). RNA extraction using Boom extraction method, and screening for SV using real-time PCR were done in the lab. Positive samples were subjected to conventional RT-PCR targeting the capsid fragment. Positive sample isolates were genotyped using Sanger sequencing. Overall SV were detected in 14.1% (40/284) of the stool samples (16.7% (38/228) of diarrhoeal and 3.6% (2/56) of non-diarrhoeal samples). Significant correlation between SV positive cases and water sources was noted. Genogroup-I was identified as the most prevalent strain comprising 81.3% (13/16), followed by SV-GII 12.5% (2/16) and SV-GIV 6.2% (1/16). This study provides valuable data on prevalence of SV amongst outpatients in rural and underdeveloped communities, and highlights the necessity for further monitoring of SV circulating strains as potential emerging strains.

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