scholarly journals Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aravind Natarajan ◽  
Alvin Han ◽  
Soumaya Zlitni ◽  
Erin F. Brooks ◽  
Summer E. Vance ◽  
...  
Keyword(s):  
Respiratory Infection ◽  
Viral Rna ◽  
Viral Pathogen ◽  
Patient Health ◽  
Reliable Quantification

AbstractPatients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health, epidemiology, and diagnosis. However, methods to preserve stool, and to extract and quantify viral RNA are not standardized. We test the performance of three preservative approaches at yielding detectable SARS-CoV-2 RNA: the OMNIgene-GUT kit, Zymo DNA/RNA shield kit, and the most commonly applied, storage without preservative. We test these in combination with three extraction kits: QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. We also test the utility of ddPCR and RT-qPCR for the reliable quantification of SARS-CoV-2 RNA from stool. We identify that the Zymo DNA/RNA preservative and the QiaAMP extraction kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR. Taken together, we recommend a comprehensive methodology for preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

scholarly journals Standardized and optimized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

2021 ◽  
Author(s):  
Aravind Natarajan ◽  
Alvin Han ◽  
Soumaya Zlitni ◽  
Erin F Brooks ◽  
Summer E Vance ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Viral Rna ◽  
Detection Methods ◽  
Common Condition ◽  
Viral Pathogen ◽  
Standardized Protocol ◽  
Patient Health ◽  
Stool Samples ◽  
Key Features ◽  
Rna Concentration

COVID-19 patients shed SARS-CoV-2 viral RNA in their stool, sometimes well after they have cleared their respiratory infection. This feature of the disease may be significant for patient health, epidemiology, and diagnosis. However, to date, methods to preserve stool samples from COVID patients, and to extract and quantify viral RNA concentration have yet to be optimized. We sought to meet this urgent need by developing and benchmarking a standardized protocol for the fecal detection of SARS-CoV-2 RNA. We test three preservative conditions for their ability to yield detectable SARS-CoV-2 RNA: OMNIgene-GUT, Zymo DNA/RNA shield kit, and the most common condition, storage without any preservative. We test these in combination with three extraction kits: the QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. Finally, we also test the utility of two detection methods, ddPCR and RT-qPCR, for the robust quantification of SARS-CoV-2 viral RNA from stool. We identify that the Zymo DNA/RNA shield collection kit and the QiaAMP viral RNA mini kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR assays. We also demonstrate key features of experimental design including the incorporation of appropriate controls and data analysis, and apply these techniques to effectively extract viral RNA from fecal samples acquired from COVID-19 outpatients enrolled in a clinical trial. Finally, we recommend a comprehensive methodology for future preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

scholarly journals Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

2021 ◽  
Author(s):  
Aravind Natarajan ◽  
Alvin Han ◽  
Soumaya Zlitni ◽  
Erin F. Brooks ◽  
Summer E. Vance ◽  
...  
Keyword(s):  
Respiratory Infection ◽  
Fecal Sample ◽  
Rna Extraction ◽  
Viral Rna ◽  
Bovine Coronavirus ◽  
Standardized Protocol ◽  
Specialized Equipment ◽  
Ease Of Access

Abstract COVID-19 patients shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. In our benchmarking study, we recommend a standardized protocol for the preservation, extraction and detection of viral RNA from stool. This protocol includes a preservative, viral RNA extraction steps, and PCR-based quantification methods to maximize yield and detection of SARS-CoV-2 RNA. Our protocol takes advantage of commercially available reagents and equipment to maximize ease of access and consistency across studies. Additionally, we apply an attenuated bovine coronavirus vaccine as a spike-in control, and synthetic RNA standards to improve standardization and reliability of the assay. While we recommend both ddPCR and RT-qPCR-based assays, we acknowledge that ddPCR may be prohibitively expensive due to the necessity of specialized equipment and reagents. This protocol was developed with a focus on SARS-CoV-2 RNA, but may apply to other coronaviruses as well. We estimate that this protocol takes between 6 to 8 hours total to quantify the viral RNA load in a fecal sample.

scholarly journals Identifying Initiation and Elongation Inhibitors of Dengue Virus RNA Polymerase in a High-Throughput Lead-Finding Campaign

2014 ◽  
Vol 20 (1) ◽  
pp. 153-163 ◽  
Author(s):  
Thomas M. Smith ◽  
Siew Pheng Lim ◽  
Kimberley Yue ◽  
Scott A. Busby ◽  
Rishi Arora ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
High Throughput ◽  
Rna Synthesis ◽  
De Novo ◽  
Antiviral Drug ◽  
Viral Rna ◽  
Renilla Luciferase ◽  
Viral Pathogen ◽  
Open Conformation

Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a “closed” to “open” conformation to accommodate the viral RNA template. Inhibitors that lock the “closed” or block the “open” conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography–mass spectrometry. The ability of inhibitors to bind and stabilize a “closed” conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase–based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.

scholarly journals Respiratory Infections and Activity of Anti-Flu Vaccine in Patients with Lymphoma and Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4617-4617
Author(s):  
Noa Lavi ◽  
Emilia Hardak ◽  
Ilana Oren ◽  
Irit Avivi
Keyword(s):  
Multiple Myeloma ◽  
Cancer Therapy ◽  
Respiratory Infection ◽  
Viral Infections ◽  
Respiratory Infections ◽  
Viral Pathogen ◽  
Flu Vaccination ◽  
Anti Cancer ◽  
Flu Vaccine ◽  
Tract Infections

Abstract Introduction: The significance of viral respiratory infections (VRI) and efficacy of anti-flu vaccine in patients (pts) with lymphoproliferative malignancies (LPM) have not been fully elucidated. The current large comprehensive study was designed to evaluate the incidence of respiratory infections in LPM pts, define clinical significance of VRI and assess the impact of anti-flu vaccine on RI prevention in this population. Methods: All consecutive pts, diagnosed either with non-Hodgkin lymphoma (NHL) or multiple myeloma (MM), followed at the Rambam Hematology Outpatient Unit between 01/2011 and 03/2012 (during or after anti-cancer therapy or untreated) were evaluated. Data regarding anti-lymphoma/MM therapy used, respiratory infections occurring during the study period, tests performed to assess these infections (focusing on viral ones), administration of anti-flu vaccine and infection-related outcome were recorded. Samples of nasopharyngeal aspirate (NPA) or bronchoalveolar lavage (BAL) were evaluated for VRI pathogens using both immunofluorescence and polymerase chain reaction (PCR) methods. Results: Five hundred and sixty two respiratory infection episodes were reported in 369 pts. In 224 (40%) episodes, VRI investigation was performed using either NPA (n=180) or BAL (n=44) samples. The decision regarding the sample source was taken based on the respiratory infection severity. The cohort screened for VRI included a larger proportion of pts receiving anti-cancer therapy during the study period than the non-screened cohort (70% vs 30%, respectively, p<0.01). The number of respiratory episodes requiring hospitalization for their respiratory infection was higher in the former cohort (54% vs 18%). One hundred fifty two screened episodes were community-acquired (50 required hospitalization) and 72 were hospital-acquired. Fever was the most common clinical symptom in screened episodes (n=111, 50%), followed by cough (n=91, 41%), dyspnea (n= 87, 39%), runny nose (n=47, 21%), respiratory distress (n=45, 20%): with decreased O2 saturation (n=35, 16%) and mechanical ventilation (n=10, 4%). In 12% and 23% of episodes, respectively, neutropenia and significant hypogammaglobinemia (IgG <500 mg/dl) were revealed. VRI were identified in 66/224 episodes (29%), 59 episodes were viral infections and 7 - co-infections (viral with another pathogen). Sixty viral episodes were diagnosed in pts with upper respiratory tract infections and 6 in pts with pneumonia. Cough, abnormal lung physical findings and hypogammaglobinemia were statistically more frequent in pts with viral infections vs those with other respiratory infections. Viral infections were treated according to pathogens recognized. Tamiflu was prescribed for influenza infections and ribavirin was given for part of the respiratory syncytial virus (RSV) infections. Supportive treatment only was given for parainfluensa and metapneumovirus infections. Twelve episodes of VRI, developed outside of hospital, required hospitalization (5%) and 2 of these episodes resulted in death. The most frequent viral infection was Influenza A (22 cases), followed by RSV (14 cases), parainfluenza and metapneumovirus (11 cases each). Less frequent viral pathogens were Influenza B (6 cases), and adenovirus (2 cases). Seven cases (10%) were accompanied with a secondary bacterial or fungal infection. Notably, 3 of the 10 pts diagnosed with RSV infection had a concurrent Aspergillus infection. Data regarding anti-flu vaccination were available in 701 pts; 431 pts (61%) were vaccinated. In a univariate analysis, anti-flu vaccination was found to have no impact on the respiratory infection frequency (51% vs 48.9%, p=0.752). Among pts with VRI, 49% did and 45% did not previously receive anti-flu vaccination (p=0.95). This vaccination had no effect on the VRI incidence in the investigated episodes. Conclusions: VRI, although frequent, are usually associated with a good outcome in LPM pts. Presence of cough and abnormal pulmonary examination were found to predict a positive screen for a viral pathogen. Influenza and RSV, both potentially treatable with Tamiflu and ribavirin, respectively, were responsible for 55% of VRI episodes, justifying a wider use of viral screening in LPM pts. Anti-flu vaccination has not proved its efficacy either in preventing viral infections or in reducing the entire incidence of respiratory infections in these pts. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of a Natural Viral RNA Motif That Optimizes Sensing of Viral RNA by RIG-I

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Jie Xu ◽  
Xiomara Mercado-López ◽  
Jennifer T. Grier ◽  
Won-keun Kim ◽  
Lauren F. Chun ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Antiviral Immunity ◽  
Antiviral Response ◽  
Viral Rna ◽  
Immune Recognition ◽  
Immunostimulatory Activity ◽  
Viral Pathogen ◽  
Double Stranded Rna ◽  
Rna Motif ◽  
Viral Recognition

ABSTRACTStimulation of the antiviral response depends on the sensing of viral pathogen-associated molecular patterns (PAMPs) by specialized cellular proteins. During infection with RNA viruses, 5′-di- or -triphosphates accompanying specific single or double-stranded RNA motifs trigger signaling of intracellular RIG-I-like receptors (RLRs) and initiate the antiviral response. Although these molecular signatures are present during the replication of many viruses, it is unknown whether they are sufficient for strong activation of RLRs during infection. Immunostimulatory defective viral genomes (iDVGs) from Sendai virus (SeV) are among the most potent natural viral triggers of antiviral immunity. Here we describe an RNA motif (DVG70-114) that is essential for the potent immunostimulatory activity of 5′-triphosphate-containing SeV iDVGs. DVG70-114enhances viral sensing by the host cell independently of the long stretches of complementary RNA flanking the iDVGs, and it retains its stimulatory potential when transferred to otherwise inert viral RNA.In vitroanalysis showed that DVG70-114augments the binding of RIG-I to viral RNA and promotes enhanced RIG-I polymerization, thereby facilitating the onset of the antiviral response. Together, our results define a new natural viral PAMP enhancer motif that promotes viral recognition by RLRs and confers potent immunostimulatory activity to viral RNA.IMPORTANCEA discrete group of molecular motifs, including 5′-triphosphates associated with double-stranded RNA, have been identified as essential for the triggering of antiviral immunity. Most RNA viruses expose these motifs during their replication; however, successful viruses normally evade immune recognition and replicate to high levels before detection, indicating that unknown factors drive antiviral immunity. DVGs from SeV are among the most potent natural viral stimuli of the antiviral response known to date. These studies define a new natural viral motif present in DVGs that maximizes viral recognition by the intracellular sensor RIG-I, allowing fast and strong antiviral responses even in the presence of viral-encoded immune antagonists. This motif can be harnessed to increase the immunostimulatory potential of otherwise inert viral RNAs and represents a novel immunostimulatory enhancer that could be used in the development of vaccine adjuvants and antivirals.

Diagnosis of Atypical Pathogens in Patients Hospitalized with Community-acquired Respiratory Infection

2004 ◽  
Vol 36 (4) ◽  
pp. 269-273 ◽  
Author(s):  
Peter M. Schneeberger ◽  
J. Wendeline Dorigo-zetsma ◽  
Anneke van der Zee ◽  
Marion van Bon ◽  
Jean-louis van Opstal
Keyword(s):  
Respiratory Infection ◽  
Atypical Pathogens

Somatoforme Symptome, Angst und Depression bei Migranten aus der Türkei, aus Osteuropa und aus der ehemaligen Sowjetunion

2010 ◽  
Vol 58 (3) ◽  
pp. 165-171 ◽  
Author(s):  
Ricarda Mewes ◽  
Winfried Rief ◽  
Alexandra Martin ◽  
Heide Glaesmer ◽  
Elmar Brähler
Keyword(s):  
Psychische Gesundheit ◽  
Patient Health Questionnaire ◽  
Health Questionnaire ◽  
Epidemiologische Studien ◽  
Patient Health

Hintergrund: Gegenüberstellende epidemiologische Studien zur psychischen Gesundheit bei verschiedenen Migrantengruppen in Deutschland fehlen weitgehend. Sie sind jedoch von großer Wichtigkeit, um den Therapiebedarf für diese Gruppen zu bestimmen und Angebote entsprechend auszurichten. Die vorliegende Studie möchte die Ausprägung einer depressiven, somatoformen und angstbezogenen Symptomatik bei osteuropäischen, türkischen und Migranten aus der ehemaligen Sowjetunion in der Allgemeinbevölkerung miteinander vergleichen und untersuchen, ob es Unterschiede in der Bereitschaft gibt, für verschiedene Beschwerden einen Arzt aufzusuchen. Methode: 43 osteuropäische Migranten (beide Eltern in Polen, Rumänien, Slowakischer Republik, Tschechischer Republik oder Ungarn geboren), 49 Migranten aus der ehemaligen Sowjetunion (beide Eltern in Russland, Ukraine, Weißrussland oder Kasachstan geboren; Russlanddeutsche Personen fallen auch in diese Gruppe) und 42 Personen mit türkischem Migrationshintergrund wurden mit dem Patient-Health-Questionnaire auf depressive, somatoforme und angstbezogene Symptome untersucht und mit einem Fragebogen zu ihrer Bereitschaft befragt, für verschiedene Beschwerden einen Arzt aufzusuchen. Die Gruppen wurden mit Varianzanalysen unter Kontrolle möglicher konfundierender Variablen miteinander verglichen. Ergebnisse: Unter Kontrolle von Geschlecht, Alter, Partnerschaft und Erwerbstätigkeit zeigten sich keine signifikanten Unterschiede in depressiver, somatoformer und ängstlicher Symptomatik zwischen den drei Gruppen. Unter Kontrolle für Alter und Geschlecht zeigten sich ebenfalls keine Unterschiede in der Bereitschaft, für verschiedene Beschwerden einen Arzt aufzusuchen. Frauen berichteten mehr somatoforme Beschwerden als Männer und zeigten eine höhere Bereitschaft, einen Arzt zu konsultieren. Diskussion: Es lassen sich keine kulturellen Einflüsse in Bezug auf die psychische Gesundheit und den Umgang mit verschiedenen Beschwerden bei diesen eher gut integrierten Migranten feststellen. Weitere Studien zu dieser Fragestellung mit größeren Stichproben und unter Einbezug schlechter Deutsch sprechender Migranten wären wünschenswert.

Beurteilung depressiver und somatischer Symptome mittels des PHQ-9 und PHQ-15 bei ambulanten vietnamesischen und deutschen Patientinnen

2016 ◽  
Vol 64 (1) ◽  
pp. 25-36 ◽  
Author(s):  
Eric Hahn ◽  
Ronald Burian ◽  
Annegret Dreher ◽  
Georg Schomerus ◽  
Michael Dettling ◽  
...  
Keyword(s):  
Patient Health Questionnaire ◽  
Health Questionnaire ◽  
Fand Sich ◽  
Patient Health ◽  
Somatische Symptome ◽  
Vergleichende Untersuchungen

Zusammenfassung. Studien in der Allgemeinbevölkerung zeigen uneinheitliche Ergebnisse bezüglich häufigerer Somatisierung bei Migranten. Vergleichende Untersuchungen fanden bei depressiven Patienten ostasiatischer Herkunft geringere Angaben von psychologischen Symptomen und häufigere somatische Beschwerden, als bei Patienten westlicher Herkunft. Aufgrund einer geringen Inanspruchnahme psychiatrischer Versorgungsangebote in Deutschland, insbesondere durch vietnamesische Migranten der ersten Generation, existieren bisher keine Studien zu einer psychischen und somatischen Symptomausprägung bei Patienten vietnamesischer Herkunft im Vergleich zu deutschen Patienten ohne Migrationshintergrund. Im Kontext kultursensibler Diagnostik von Migranten in Deutschland wurde als ausreichend messäquivalentes Selbstbeurteilungsinstrument insbesondere der Patient Health Questionnaire bzw. der Gesundheitsfragebogen für Patienten als ein valides und einfach verwendbares Instrument für eine Erfassung von Symptomen und Schweregraden häufiger psychischer Störungen, wie der Depression empfohlen. Im Rahmen der vorliegenden Untersuchung wurden bei 66 vietnamesischen Patientinnen der ersten Generation und 83 deutschen Patientinnen während des erstmaligen psychiatrischen Kontaktes psychische Symptome einer Depression mittels des PHQ-9 und somatische Symptome mittels des PHQ-15 in der jeweiligen Muttersprache erfasst. Für beide Gruppen fand sich für beide Instrumente eine zufriedenstellende interne Konsistenz. Ein möglicher Zusammenhang zwischen der Herkunft und dem Schweregrad der Ausprägung psychischer depressiver und somatischer Symptome bei diagnostizierter depressiver Episode erfolgte mittels einer multivariaten Analyse. Für die Selbstbeurteilung mittels des PHQ-9 fanden sich keine Gruppenunterschiede hinsichtlich des Gesamtsummenwertes und des Schweregrades psychischer depressiver Symptome. Dagegen berichteten vietnamesische Patientinnen in der Selbstwahrnehmung anhand des PHQ-15 von einem insgesamt höheren Schweregrad von somatischen Symptomen. Insbesondere waren bei depressiven vietnamesischen Patientinnen die Mittelwerte der Einzelitems Kopfschmerzen, Glieder- und Gelenkschmerzen, Schmerzen im Brustbereich sowie Schwindel und Ohnmachtsanfälle gegenüber deutschen Patientinnen deutlich erhöht. Entgegen der Untersuchungshypothese und früherer Studien ging die häufigere Selbstbeurteilung oder Aufmerksamkeit auf somatische Symptome bei vietnamesischen Patientinnen nicht mit einer verminderten Eigenwahrnehmung von psychischen Symptomen einer depressiven Episode anhand des PHQ-9 einher.

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