A Robust, Safe and Scalable Magnetic Nanoparticle Workflow for RNA Extraction of Pathogens from Clinical and Environmental Samples

ABSTRACTDiagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magnetic-nanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. Using 36 COVID-19 patient samples, 2 wastewater samples and 1 human pathogens control sample, we showed that MAVRICS rivals commercial kits in validated diagnostic tests of SARS-CoV-2, influenza viruses, and respiratory syncytial virus. MAVRICS is scalable and thus could become an enabling technology for widespread community testing and wastewater monitoring in the current and future pandemics.

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MAVRICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples v2

10.17504/protocols.io.b2p8qdrw ◽

2021 ◽

Author(s):

Mo Li ◽

Gerardo Ramos-Mandujano

Keyword(s):

Extraction Method ◽

Magnetic Nanoparticle ◽

Rna Extraction ◽

Rna Isolation ◽

Emerging Pathogens ◽

Nucleic Acid Isolation ◽

Guanidinium Thiocyanate ◽

Biology Laboratory ◽

Alternative Solutions ◽

Basic Equipment

Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magneticnanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities.

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Spring is coming, where are the Respiratory Syncytial Virus and Influenza viruses?

Journal of Clinical Virology ◽

10.1016/j.jcv.2021.104824 ◽

2021 ◽

pp. 104824

Author(s):

Eric Farfour ◽

Tiffany Pascreau ◽

Emilie Jolly ◽

Sara Zia-Chahabi ◽

Laurence Mazaux ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Influenza Viruses ◽

Syncytial Virus

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Thapsigargin Is a Broad-Spectrum Inhibitor of Major Human Respiratory Viruses: Coronavirus, Respiratory Syncytial Virus and Influenza A Virus

Viruses ◽

10.3390/v13020234 ◽

2021 ◽

Vol 13 (2) ◽

pp. 234

Author(s):

Sarah Al-Beltagi ◽

Cristian Alexandru Preda ◽

Leah V. Goulding ◽

Joe James ◽

Juan Pu ◽

...

Keyword(s):

Influenza Virus ◽

Respiratory Syncytial Virus ◽

Influenza A Virus ◽

Broad Spectrum ◽

Influenza A ◽

Respiratory Viruses ◽

Influenza Viruses ◽

Virus Challenge ◽

2009 H1n1 ◽

Syncytial Virus

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG’s antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.

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Where future emerging pathogens will come from and what approaches can be used to find them, besides VFARs

Journal of Water and Health ◽

10.2166/wh.2009.096 ◽

2009 ◽

Vol 7 (S1) ◽

pp. S75-S93 ◽

Cited By ~ 8

Author(s):

Mark D. Sobsey ◽

Suresh D. Pillai

Keyword(s):

Human Health ◽

Antibiotic Use ◽

Epidemiological Surveillance ◽

Waterborne Pathogens ◽

Human Pathogens ◽

Emerging Pathogens ◽

Antibiotic Resistant ◽

Waterborne Pathogen ◽

Pathogen Virulence ◽

Human Health Effects

A consideration of available evidence for some known and well-characterized waterborne pathogens suggests that the diversity of pathogen virulence mechanisms and properties is too great to specifically predict the emergence and future human health impacts of new waterborne pathogens. However, some future emerging pathogens are existing microbes that will be discovered to cause disease. Some will arise from existing ones by either predictable evolutionary and adaptation changes or by unpredictable changes involving a variety of biotic and abiotic mechanisms. Many, and perhaps most, emerging waterborne human pathogens will be zoonotic agents or come from other non-human reservoirs. The emergence of some waterborne pathogens will be related to antibiotic use, resulting in emerging antibiotic-resistant waterborne pathogens. Reliably predicting pathogen emergence and human health effects based on VFARs or other properties of microbes and their hosts is not possible at this time. This is because of (1) the diversity of microbes and their virulence and pathogenicity properties, (2) their ability to change unpredictably, (3) their intimate and diverse interrelationships with a myriad of hosts and dynamic natural and anthropogenic environments and (4) the subtle variations in the immune status of individuals. The best available approach to predicting waterborne pathogen emergence is through vigilant use of microbial, infectious disease and epidemiological surveillance. Understanding the microbial metagenome of the human body can also lead to a better understanding of how we define and characterize pathogens, commensals and opportunists.

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Influenza C and D viral load in cattle correlates with bovine respiratory disease (BRD): Emerging role of orthomyxoviruses in the pathogenesis of BRD

10.1101/2020.05.26.118166 ◽

2020 ◽

Author(s):

Ruth H. Nissly ◽

Noriza Zaman ◽

Puteri Ainaa S. Ibrahim ◽

Kaitlin McDaniel ◽

Levina Lim ◽

...

Keyword(s):

Respiratory Disease ◽

Geometric Mean ◽

Bovine Respiratory Disease ◽

The United States ◽

Influenza Viruses ◽

Cattle Industry ◽

Diarrhea Virus ◽

Viral Loads ◽

Influenza C Virus ◽

Syncytial Virus

AbstractBovine respiratory disease (BRD) is the costliest disease affecting the cattle industry globally. Despite decades of research, the pathophysiology of BRD is not yet fully understood. It is widely believed that viruses predispose cattle to bacterial infection by causing direct damage to the respiratory tract and interfering with the immune system, leading to bacterial pneumonia. BRD remains a major challenge despite extensive vaccination against all major viral pathogens associated with the disease. Orthomyxoviruses (Influenza C & D viruses), have recently been found to infect cattle throughout the United States and are implicated to play a role in BRD. Here, we use the largest cohort study to date to investigate the association of influenza viruses in cattle with BRD. Cattle (n=599) from 3 locations were individually observed and scored for respiratory symptoms using the McGuirk scoring system. Deep pharyngeal and mid-nasal swabs were collected from each animal and were tested quantitatively for bovine viral diarrhea virus, bovine herpesvirus 1, bovine respiratory syncytial virus, bovine coronavirus, influenza C virus (ICV) and influenza D virus (IDV) by real-time PCR. Cattle that have higher viral loads of IDV and ICV also have greater numbers of co-infecting viruses than controls. More strikingly, in BRD-symptomatic cattle, the geometric mean of detectable IDV viral RNA was nearly 2 logs higher in co-infected animals (1.30×104) than those singly infected with IDV (2.19×102). This is strong evidence that viral coinfections can lead to higher replication of IDV. Our results strongly suggest that orthomyxoviruses may be significant contributors to BRD.

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Circulation of Respiratory Viruses in Hospitalized Adults before and during the COVID-19 Pandemic in Brescia, Italy: A Retrospective Study

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18189525 ◽

2021 ◽

Vol 18 (18) ◽

pp. 9525

Author(s):

Maria Antonia De Francesco ◽

Caterina Pollara ◽

Franco Gargiulo ◽

Mauro Giacomelli ◽

Arnaldo Caruso

Keyword(s):

Respiratory Syncytial Virus ◽

Influenza A ◽

Statistical Significance ◽

Respiratory Viruses ◽

Influenza Viruses ◽

Contact Tracing ◽

Nucleic Acid Detection ◽

Respiratory Pathogens ◽

Syncytial Virus ◽

Human Coronaviruses

Different preventive public health measures were adopted globally to limit the spread of SARS-CoV-2, such as hand hygiene and the use of masks, travel restrictions, social distance actions such as the closure of schools and workplaces, case and contact tracing, quarantine and lockdown. These measures, in particular physical distancing and the use of masks, might have contributed to containing the spread of other respiratory viruses that occurs principally by contact and droplet routes. The aim of this study was to evaluate the prevalence of different respiratory viruses (influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, 3 and 4, rhinovirus, adenovirus, metapneumovirus and human coronaviruses) after one year of the pandemic. Furthermore, another aim was to evaluate the possible impact of these non-pharmaceutical measures on the circulation of seasonal respiratory viruses. This single center study was conducted between January 2017–February 2020 (pre-pandemic period) and March 2020–May 2021 (pandemic period). All adults >18 years with respiratory symptoms and tested for respiratory pathogens were included in the study. Nucleic acid detection of all respiratory viruses was performed by multiplex real time PCR. Our results show that the test positivity for influenza A and B, metapneumovirus, parainfluenza virus, respiratory syncytial virus and human coronaviruses decreased with statistical significance during the pandemic. Contrary to this, for adenovirus the decrease was not statistically significant. Conversely, a statistically significant increase was detected for rhinovirus. Coinfections between different respiratory viruses were observed during the pre-pandemic period, while the only coinfection detected during pandemic was between SARS-CoV-2 and rhinovirus. To understand how the preventive strategies against SARS-CoV-2 might alter the transmission dynamics and epidemic patterns of respiratory viruses is fundamental to guide future preventive recommendations.

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Simultaneous Determination of Seven Antibiotics and Five of Their Metabolites in Municipal Wastewater and Evaluation of Their Stability under Laboratory Conditions

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph182010640 ◽

2021 ◽

Vol 18 (20) ◽

pp. 10640

Author(s):

Sheng Han ◽

Xinyue Li ◽

Hongmei Huang ◽

Ting Wang ◽

Zhenglu Wang ◽

...

Keyword(s):

Public Health ◽

High Performance ◽

Municipal Wastewater ◽

Therapeutic Potential ◽

Solid Phase ◽

Human Pathogens ◽

Removal Rates ◽

Liquid Chromatography Tandem Mass ◽

Antibiotics Use ◽

Wastewater Samples

The selection and spread of antibiotic resistance poses risks to public health by reducing the therapeutic potential of antibiotics against human pathogens. Wastewater-based epidemiology (WBE) is potentially the most reliable approach to estimate antibiotics use. Previous WBE studies used parent antibiotics as biomarkers, which may lead to overestimation since parent antibiotics may be directly disposed of. Using metabolites as biomarkers can avoid this drawback. This study developed a simultaneous solid-phase extraction coupled with ultra-high-performance liquid chromatography tandem mass spectrometry method for analyzing 12 antibiotics and human metabolites in wastewater to help assess health risk. Optimum conditions were achieved using a PEP cartridge at pH 3.0. The extraction efficiencies were 73.3~95.4% in influent and 72.0~102.7% in effluent for most of the target analytes. Method detection limit ranged from 0.1 to 1.5 ng/L for influent wastewater and 0.03 to 0.7 ng/L for effluent wastewater. A stability experiment showed that sulfonamide parents and their metabolites were stable at 4 °C, −20 °C and −80 °C, while macrolides metabolites were more stable than their corresponding parents at 4 °C and −20 °C. Finally, the method was applied to measure these analytes in wastewater samples collected from three Beijing WWTPs and to derive apparent removal rates. All metabolites were detected in wastewater samples with concentrations ranging from 1.2 to 772.2 ng/L in influent, from <MDL to 235.6 ng/L in effluent. The apparent removal rates of five metabolites were above 72.6%. These results set a solid foundation for applying WBE to evaluate antibiotics use and its public health effects.

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Methods for Molecular Surveillance of Influenza Used in Macedonia

PRILOZI ◽

10.2478/prilozi-2014-0003 ◽

2014 ◽

Vol 35 (2) ◽

pp. 25-30

Author(s):

Golubinka Bosevska ◽

Elizabeta Janceska ◽

Gordana Kuzmanovska ◽

Vladimir Mikik ◽

Nikola Panovski

Keyword(s):

Influenza Virus ◽

Predictive Value ◽

Influenza A ◽

Rna Extraction ◽

Influenza Virus Infection ◽

Laboratory Diagnosis ◽

Influenza Viruses ◽

Rt Pcr ◽

Two Samples ◽

Nose And Throat

AbstractThe aim: To present and compare different Nucleic Acid Testing assays used for laboratory diagnosis of influenza virus infection in our country.Materials and methods: Respiratory samples used were nose and throat swabs. The RNA extraction was performed with a QIAamp viral RNA kit. During the season 2009–2010 the first 25 samples were tested with: conventional gel-based RT-PCR and CDC rtRT-PCR using published specific matrix and HA gene primers and probes for influenza virus typing and subtyping.Results: Of 25 samples tested with conventional RT-PCR7(28%) were positive for influenza A, but negative for A/H1seasonal and A/H3. Retested with rtRT-PCR 9(36%) were positive for influenza A, 8(32%) were positive for A/H1pdm and 1(4%) was A/H3. Two samples positive with rtRT-PCR for influenza A were negative with RT-PCR. The sensitivity of the RT-PCR in comparison with rtRT-PCR is 100% and the specificity is 88.89%. Positive predictive value for RT-PCR is 77.78%, and negative predictive value is 100%. RT-PCR is a four-step and rtRT-PCR a one-step procedure. The turn-around time of RT-PCR is 6 hours and for rtRT-PCR it is 2 hours.Discussion and conclusion: For surveillance purposes nose and throat swabs are the more easy and practical to collect. It was proved that RT-PCR is too laborious, multi-step and time-consuming. The sensitivity of both assays is equal. The specificity of rtRT-PCR is higher. NAT assays for detection of influenza viruses have become an integral component of the surveillance programme in our country. They provide a fast, accurate and sensitive detection of influenza.

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Enhancing microbiological safety of water and food

10.32469/10355/48222 ◽

2014 ◽

Author(s):

◽

Zhenyu Shen

Keyword(s):

16S Rdna ◽

In Silico ◽

Mung Bean ◽

Disease Outbreaks ◽

Laboratory Strain ◽

Human Pathogens ◽

University Of Missouri ◽

Enterohaemorrhagic Escherichia Coli ◽

Wastewater Samples

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Human and animal feces are the main sources of pathogens associated with foodborne and waterborne disease outbreaks. The studies in Volume I proposed ribosomal intervening sequences (IVSs) can be used as host-specific genetic markers for tracking fecal sources in polluted food and water. In Chapter 3, using bioinformatics tools, one IVS in Faecalibacterium 16S rDNA specific to poultry (chicken and turkey) was identified in silico. A PCR assay was then developed to confirm the IVS was only present in fecal or wastewater samples from poultry. The study in Chapter 4 comprehensively examined host specificities of IVSs in 16S rDNA of 73 genera of fecal bacteria. A total of 13 IVSs were identified to be associated particular host sources in silico and the host-specificities of eight of them were confirmed by PCR reactions. Furthermore, a novel beef cattle-specific IVS was identified with next generation sequencing (NGS). Internalization of human pathogens in produce poses high risks to human health because the surface-sanitization method fail to inactivate the pathogens. The study in Volume II proposed to control the pathogen internalization with endophytic Bacillus subtilis strains isolated from vegetables. Twelve endophytic B. subtilis strains isolated from inner tissue of lettuce stems and mung bean seeds displayed inhibitory activity against a Salmonella enterica indicator in vitro and two of them (LCA1 and M24) showed broad inhibitory spectrums. LCA1, M24, and a laboratory strain B. subtilis 168 could internalize in mung bean sprouts and significantly reduced the internalization of S. enterica and enterohaemorrhagic Escherichia coli (EHEC).

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Respiratory tract viruses

Oxford Textbook of Medicine ◽

10.1093/med/9780198746690.003.0076_update_001 ◽

2020 ◽

pp. 723-734

Author(s):

Malik Peiris

Keyword(s):

Respiratory Tract ◽

Lower Respiratory Tract ◽

Respiratory Infections ◽

Influenza Viruses ◽

Parainfluenza Viruses ◽

Clinical Syndromes ◽

Respiratory Tract Viruses ◽

Syncytial Virus ◽

Tract Infections ◽

Viral Respiratory Infections

Viral respiratory infections, including coronavirus, rhinovirus, adenovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza viruses, and influenza viruses, are a substantial cause of morbidity and mortality worldwide, most notably the COVID-19 pandemic. Transmission occurs through direct contact, contaminated fomites, and large airborne droplets, with long-range transmission by small particle aerosols reported in at least some instances of influenza and severe acute respiratory syndrome. Clinical syndromes affect the upper and/or lower respiratory tract, including coryza, pharyngitis, croup, bronchiolitis, and pneumonia. Each syndrome can potentially be caused by several viruses, and each respiratory virus can be associated with different clinical syndromes. Measles is a major cause of lower respiratory tract infections and fatality in tropical countries.

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