scholarly journals DEVELOPMENT AND TESTING OF A LOW-COST INACTIVATION BUFFER THAT ALLOWS DIRECT SARS-COV-2 DETECTION IN SALIVA

2021 ◽  
Author(s):  
Brandon Bustos-Garcia ◽  
Sylvia Garza-Manero ◽  
Nallely Cano-Dominguez ◽  
Dulce Maria Lopez Sanchez ◽  
Gonzalo Salgado-Montes de Oca ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Heat Inactivation ◽  
Independent Manner ◽  
Resource Limited ◽  
Multiplex Reaction ◽  
The Us ◽  
Diagnostic Standards ◽  
Control And Prevention ◽  
Commercial Kits

Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places where good vaccination coverage has been achieved. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing implementation in resource-limited settings. It is therefore important to develop testing protocols that facilitate implementation and are inexpensive, fast, and sufficiently sensitive. In this work, we optimized the composition of a buffer (PKTP) containing a protease, a detergent, and an RNase inhibitor, that is compatible with the RT-qPCR chemistry, allowing for direct testing of SARS-CoV-2 from saliva in an RNA extraction-independent manner. This buffer is compatible with heat-inactivation reducing the biohazard risk of handling the samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (82.2% for the N1 and 84.4% for the N2 target, respectively) and correlations (R=0.77, p<0.001 for N1, and R=0.78, p<0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to reduce the number of PCR reactions per patient and further reduce overall costs while maintaining diagnostic standards in favor of massive testing.

scholarly journals Direct Viral RNA Detection of SARS-CoV-2 and DENV in Inactivated Samples by Real-Time RT-qPCR: Implications for Diagnosis in Resource Limited Settings with Flavivirus Co-Circulation

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1558
Author(s):  
Zhan Qiu Mao ◽  
Mizuki Fukuta ◽  
Jean Claude Balingit ◽  
Thi Thanh Ngan Nguyen ◽  
Co Thach Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Early Stage ◽  
Rna Extraction ◽  
Viral Rna ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Resource Limited Settings ◽  
Rna Detection ◽  
Resource Limited ◽  
Starting Point

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

scholarly journals A global proficiency testing programme for Xpert® MTB/RIF using dried tube specimens, 2013–2015

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Katherine Klein ◽  
Kyle DeGruy ◽  
Zilma Rey ◽  
Patricia Hall ◽  
Andrea Kim ◽  
...  
Keyword(s):  
Proficiency Testing ◽  
Low Cost ◽  
The United States ◽  
Rapid Expansion ◽  
Test Results ◽  
Resource Limited ◽  
Quality Assurance Measure ◽  
Control And Prevention ◽  
Feasible Option ◽  
Important Quality

Background: Proficiency testing (PT) is an important quality assurance measure toward ensuring accurate and reliable diagnostic test results from clinical and public health laboratories. Despite the rapid expansion of the Xpert® MTB/RIF assay for the detection of tuberculosis in resource-limited settings (RLS), low-cost PT materials for Xpert MTB/RIF external quality assessment (EQA) are not widely available.Objective: We sought to determine whether a dried tube specimen (DTS)-based PT programme would be a feasible option to support Xpert MTB/RIF EQA in RLS.Methods: Between 2013 and 2015, the United States Centers for Disease Control and Prevention developed and conducted a voluntary EQA programme using DTS-based PT material. Eight rounds of PT, each comprising five DTS samples, were provided to enrolled testing sites. After each round, participant results were compared to expected results, scored as satisfactory or unsatisfactory, and sites were provided with performance reports.Results: Programme enrolment increased from 102 testing sites in seven countries to 441 testing sites in 14 countries over the course of three years. In each PT round, approximately 90% of participating sites demonstrated satisfactory performance. In seven of the 14 enrolled countries, the proportion of sites with a satisfactory score increased between the first round of participation and the most recent round of participation.Conclusion: This programme demonstrated that it is possible to implement an Xpert MTB/RIF PT programme for RLS using DTS, that substantial demand for Xpert MTB/RIF PT material exists in RLS, and that country performance can improve in a DTS-based PT programme.

scholarly journals A simple and rapid method for isolating high-quality RNA from kenaf containing high polysaccharide and polyphenol contents

2020 ◽  
Author(s):  
Xiaofang Liao ◽  
Hongwei Li ◽  
Aziz Khan ◽  
Yanhong Zhao ◽  
Wenhuan Hou ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Cost Effective ◽  
Spectrophotometric Analysis ◽  
Rt Pcr ◽  
High Quality ◽  
Time Saving ◽  
Ctab Method ◽  
Commercial Kits

AbstractThe isolation of high-quality RNA from kenaf is crucial for genetic and molecular biology studies. However, high levels of polysaccharide and polyphenol compounds in kenaf tissues could irreversibly bind to and coprecipitate with RNA, which complicates RNA extraction. In the present study, we proposed a simplified, time-saving and low-cost extraction method for isolating high quantities of high-quality RNA from several different kenaf tissues. RNA quality was measured for yield and purity, and the proposed protocol yielded high quantities of RNA (10.1-12.9 μg/g·FW). Spectrophotometric analysis showed that A260/280 ratios of RNA samples were in the range of 2.11 to 2.13, and A260/230 ratios were in the range of 2.04-2.24, indicating that the RNA samples were free of polyphenols, polysaccharides, and protein contaminants after isolation. The method of RNA extraction presented here was superior to the conventional CTAB method in terms of RNA isolation efficiency and was more sample-adaptable and cost-effective than commercial kits. Furthermore, to confirm downstream amenability, the high-quality RNA obtained from this method was successfully used for RT-PCR, real-time RT-PCR and Northern blot analysis. We provide an efficient and low-cost method for extracting high quantities of high-quality RNA from plants that are rich in polyphenols and polysaccharides, and this method was also validated for the isolation of high-quality RNA from other plants.

scholarly journals Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247792
Author(s):  
Valeria Genoud ◽  
Martin Stortz ◽  
Ariel Waisman ◽  
Bruno G. Berardino ◽  
Paula Verneri ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Standard Technique ◽  
Proteinase K ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Nasopharyngeal Swab ◽  
Reverse Transcription Pcr ◽  
Extraction Step ◽  
Commercial Kits ◽  
Inexpensive Procedure

Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.

scholarly journals A simplified SARS-CoV-2 detection protocol for research laboratories

2020 ◽  
Author(s):  
Sean Paz ◽  
Anastasia Ritchie ◽  
Christopher Mauer ◽  
Janet D. Robishaw ◽  
massimo caputi
Keyword(s):  
Rna Extraction ◽  
Clinical Diagnostics ◽  
Laboratory Research ◽  
Health Crisis ◽  
Public Health Crisis ◽  
The Us ◽  
Human Labor ◽  
Control And Prevention ◽  
The Face ◽  
Food And Drugs Administration

Widespread testing is required to limit the current public health crisis caused by the COVID-19 pandemic. Multiple tests protocols have been authorized by the food and drugs administration under an emergency use authorization (EUA). The majority of these protocols are based on the gold-standard RT-qPCR test pioneered by the U.S. Centers for Disease Control and Prevention. However, there is still a widespread lack of testing in the US and many of the clinical diagnostics protocols require extensive human labor and materials, such as RNA extraction kits, that could face supply shortages and present biosafety concerns. Given the need to develop alternative reagents and approaches to allow nucleic-acid testing in the face of heightened demand and potential shortages, we have developed a simplified SARS-CoV-2 testing protocol adapted for its use in laboratory research with minimal molecular biology equipment and expertise. The protocol requires minimal BSL1 biosafety level precautions and facilities.

scholarly journals Comparison of 12 molecular detection assays for SARS-CoV-2

2020 ◽  
Author(s):  
Yasufumi Matsumura ◽  
Tsunehiro Shimizu ◽  
Taro Noguchi ◽  
Satoshi Nakano ◽  
Masaki Yamamoto ◽  
...  
Keyword(s):  
Confidence Interval ◽  
Internal Control ◽  
Genetic Locus ◽  
Molecular Diagnostic ◽  
Molecular Testing ◽  
First Line ◽  
Diagnostic Assays ◽  
The Us ◽  
Control And Prevention ◽  
Commercial Kits

AbstractMolecular testing for SARS-CoV-2 is the mainstay for accurate diagnosis of the infection, but the diagnostic performances of available assays have not been defined. We compared 12 molecular diagnostic assays, including 8 commercial kits using 155 respiratory samples (65 nasopharyngeal swabs, 45 oropharyngeal swabs, and 45 sputum) collected at 2 Japanese hospitals. Sixty-eight samples were positive for more than one assay and one genetic locus and were defined as true positive samples. All the assays showed a specificity of 100% (95% confidence interval, 95.8 to 100). The N2 assay kit of the US Centers for Disease Control and Prevention (CDC) and the N2 assay of the Japanese National Institute of Infectious Disease (NIID) were the most sensitive assays with 100% sensitivity (95% confidence interval, 94.7 to 100), followed by the CDC N1 kit, E assay by Corman, and NIID N2 assay multiplex with internal control reactions. These assays are reliable as first-line molecular assays in laboratories when combined with appropriate internal control reactions.

scholarly journals Inactivation of SARS Coronavirus 2 and COVID-19 patient samples for contemporary immunology and metabolomics studies

2021 ◽  
Author(s):  
Devon J. Eddins ◽  
Leda C. Bassit ◽  
Joshua Chandler ◽  
Natalie S. Haddad ◽  
Kathryn Musall ◽  
...  
Keyword(s):  
Single Cell ◽  
World Health ◽  
Antibody Detection ◽  
Heat Inactivation ◽  
Viral Inactivation ◽  
The Us ◽  
Control And Prevention ◽  
Level 3 ◽  
Infected Cells ◽  
Health Organization

In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from Wuhan, China spurring the Coronavirus Disease-19 (COVID-19) pandemic that has resulted in over 219 million confirmed cases and nearly 4.6 million deaths worldwide. Intensive research efforts ensued to constrain SARS-CoV-2 and reduce COVID-19 disease burden. Due to the severity of this disease, the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level 3 (BSL3) containment laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred and manipulated at lower containment levels (i.e., BSL2), and maintain the fidelity of downstream assays to expedite the development of medical countermeasures (MCMs). We demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with paraformaldehyde solution or other commonly-used branded reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and antibody detection assays, heat inactivation following cDNA amplification of single-cell emulsions for droplet-based single-cell mRNA sequencing applications, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of protocols for viral inactivation of SARS-CoV-2 and COVID-19 patient samples for downstream contemporary immunology assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

scholarly journals Validation of a point-of-care rapid diagnostic test for hepatitis C for use in resource-limited settings

2019 ◽  
Vol 11 (4) ◽  
pp. 314-315
Author(s):  
James S Leathers ◽  
Maria Belen Pisano ◽  
Viviana Re ◽  
Gertine van Oord ◽  
Amir Sultan ◽  
...  
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Rapid Diagnosis ◽  
Direct Acting Antivirals ◽  
Resource Limited Settings ◽  
Rapid Diagnosis Test ◽  
Resource Limited ◽  
Specificity And Sensitivity ◽  
Hcv Screening ◽  
Direct Acting

Abstract Background Treatment of HCV with direct-acting antivirals has enabled the discussion of HCV eradication worldwide. Envisioning this aim requires implementation of mass screening in resource-limited areas, usually constrained by testing costs. Methods We validated a low-cost, rapid diagnosis test (RDT) for HCV in three different continents in 141 individuals. Results The HCV RDT showed 100% specificity and sensitivity across different samples regardless of genotype or viral load (in samples with such information, 90%). Conclusions The HCV test validated in this study can allow for HCV screening in areas of need when properly used.

scholarly journals Information and information resources in COVID-19: Awareness, control, and prevention

2021 ◽  
pp. 096100062110165
Author(s):  
Mohammadhiwa Abdekhoda ◽  
Fatemeh Ranjbaran ◽  
Asghar Sattari
Keyword(s):  
Low Cost ◽  
Information Resources ◽  
Age And Gender ◽  
Use Of Resources ◽  
Strategic Approach ◽  
Wide Range ◽  
Data Collection Instrument ◽  
Control And Prevention ◽  
And Gender ◽  
And Control

This study was conducted with the aim of evaluating the role of information and information resources in the awareness, control, and prevention of COVID-19. This study was a descriptive-analytical survey in which 450 participants were selected for the study. The data collection instrument was a researcher-made questionnaire. Descriptive and inferential statistics were used to analyze the data through SPSS. The findings show that a wide range of mass media has become well known as information resources for COVID-19. Other findings indicate a significant statistical difference in the rate of using information resources during COVID-19 based on age and gender; however, this difference is not significant regarding the reliability of information resources with regard to age and gender. Health information has an undisputable role in the prevention and control of pandemic diseases such as COVID-19. Providing accurate, reliable, and evidence-based information in a timely manner for the use of resources and information channels related to COVID-19 can be a fast and low-cost strategic approach in confronting this disease.

scholarly journals Stethoscope hygiene: A call to action. Recommendations to update the CDC guidelines

2021 ◽  
pp. 1-3
Author(s):  
Sarathi Kalra ◽  
Alpesh Amin ◽  
Nancy Albert ◽  
Cindy Cadwell ◽  
Cole Edmonson ◽  
...  
Keyword(s):  
Healthcare Providers ◽  
Acoustic Properties ◽  
Patient Encounter ◽  
Call To Action ◽  
The Us ◽  
Cdc Guidelines ◽  
Control And Prevention ◽  
Healthcare Acquired Infections ◽  
Current Guidance ◽  
Tremendous Challenge

Abstract Healthcare-acquired infections are a tremendous challenge to the US medical system. Stethoscopes touch many patients, but current guidance from the Centers for Disease Control and Prevention does not support disinfection between each patient. Stethoscopes are rarely disinfected between patients by healthcare providers. When cultured, even after disinfection, stethoscopes have high rates of pathogen contamination, identical to that of unwashed hands. The consequence of these practices may bode poorly in the coronavirus 2019 disease (COVID-19) pandemic. Alternatively, the CDC recommends the use of disposable stethoscopes. However, these instruments have poor acoustic properties, and misdiagnoses have been documented. They may also serve as pathogen vectors among staff sharing them. Disposable aseptic stethoscope diaphragm barriers can provide increased safety without sacrificing stethoscope function. We recommend that the CDC consider the research regarding stethoscope hygiene and effective solutions to contemporize this guidance and elevate stethoscope hygiene to that of the hands, by requiring stethoscope disinfection or change of disposable barrier between every patient encounter.

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