scholarly journals RNA-extraction-free diagnostic method to detect SARS-CoV-2: an assessment from two States, India

2021 ◽  
Author(s):  
Madhumathi Jayaprakasam ◽  
Sumit Aggarwal ◽  
Arati Mane ◽  
Vandana Saxena ◽  
Amrita Rao ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Reference Method ◽  
Independent Study ◽  
Proteinase K ◽  
Index Method ◽  
Pressing Time ◽  
Viral Transport Medium ◽  
Increasing Demand ◽  
Study Sites ◽  
Low Sensitivity

With increasing demand for large numbers of testing during COVID-19 pandemic, came alternative protocols with shortened turn-around time. We evaluated the performance of such an approach wherein 1138 consecutive clinic attendees were enrolled; 584 and 554 respectively from two independent study sites in the cities of Pune and Kolkata. Paired nasopharyngeal and oropharyngeal swabs were tested by using both reference and index methods in blinded fashion. Prior to conducting RT-PCR, swabs collected in viral transport medium (VTM) were processed for RNA extraction (reference method) and swabs collected in dry tube without VTM were incubated in Tris-EDTA-Proteinase K buffer for 30 minutes and heat inactivated at 98oC for 6 minutes (index method). Overall sensitivity and specificity of the index method were 78.9% (95% CI 71% to 86%) and 99 % (95% CI 98% to 99.6%) respectively. Agreement between the index and reference method was 96.8 % (k = 0.83, SE=0.030). The reference method exhibited enhanced detection of viral genes (E, N and RdRP) with lower Ct values compared to the index method. The index method can be used for detecting SARS-CoV-2 infection with appropriately chosen primer-probe set and heat treatment approach in pressing time; low sensitivity constrains its potential wider use.

scholarly journals Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

2020 ◽  
Author(s):  
Julien Fassy ◽  
Caroline Lacoux ◽  
Sylvie Leroy ◽  
Latifa Noussair ◽  
Sylvain Hubac ◽  
...  
Keyword(s):  
High Throughput ◽  
Cellular Response ◽  
Rna Extraction ◽  
Reference Method ◽  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Rna Purification ◽  
Large Populations ◽  
Reaction Volumes

AbstractThe emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a couple of probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction steps. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the Biomark instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring in addition to SARS-CoV-2 probes of other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). Its 10 nL range volume is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several procedures, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

scholarly journals Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0243333
Author(s):  
Julien Fassy ◽  
Caroline Lacoux ◽  
Sylvie Leroy ◽  
Latifa Noussair ◽  
Sylvain Hubac ◽  
...  
Keyword(s):  
High Throughput ◽  
Cellular Response ◽  
Rna Extraction ◽  
Reference Method ◽  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Extraction Step ◽  
Large Populations ◽  
Reaction Volumes

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

The Diagnosis of Established Deep Vein Thrombosis

1979 ◽  
Author(s):  
R. Hull ◽  
J. Hirsh
Keyword(s):  
Deep Vein Thrombosis ◽  
Doppler Ultrasonography ◽  
Clinical Symptoms ◽  
Reference Method ◽  
Impedance Plethysmography ◽  
Vein Thrombosis ◽  
Non Invasive ◽  
Patient Morbidity ◽  
Low Sensitivity ◽  
Deep Vein

It is now generally accepted that the clinical diagnosis of deep venous thrombosis (DVT) is inaccurate both because of low sensitivity and specificity. Because more than 50% of symptomatic patients fail to show thrombi on venography, anticoagulant therapy on the basis of clinical symptoms of DVT is not acceptable. Venography has been the standard reference method for the diagnosis of DVT but is invasive and consequently associated with patient morbidity. Impedance plethysmography (IPG) and Doppler ultrasonography (Doppler) are both non-invasive and, in patients with clinically suspected DVT, are sensitive and specific tests for proximal DVT. Both tests are relatively insensitive to calf DVT. IPG has the advantage of being an objective technique whereas Doppler is subjective and its accuracy may suffer in inexperienced hands. 125I fibrinogen leg scanning (leg scanning) is an inappropriate test when used alone in patients with clinically suspected DVT as it is insensitive in the upper thigh, may be negative in 30% of patients with established DVT and may take up to 72 hours to become positive. The combination, however, of IPG and leg scanning provides an accurate approach for the detection of both proximal and calf DVT in patients with established DVT. This approach is not associated with patient morbidity and offers the clinician an alternative to venography.

scholarly journals Rapid isothermal amplification and portable detection system for SARS-CoV-2

2020 ◽  
Vol 117 (37) ◽  
pp. 22727-22735 ◽  
Author(s):  
Anurup Ganguli ◽  
Ariana Mostafa ◽  
Jacob Berger ◽  
Mehmet Y. Aydin ◽  
Fu Sun ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Alternative Pathway ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Complete Agreement ◽  
Rt Pcr ◽  
Viral Transport Medium

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

scholarly journals Sensitivity of rapid antigen testing and RT-PCR performed on nasopharyngeal swabs versus saliva samples in COVID-19 hospitalized patients: results of a prospective comparative trial (RESTART)

2021 ◽  
Author(s):  
Antonios Kritikos ◽  
Giorgia Caruana ◽  
René Brouillet ◽  
John-Paul Miroz ◽  
Abed-Maillard Samia ◽  
...  
Keyword(s):  
Comparative Trial ◽  
Hospitalized Patients ◽  
Rt Pcr ◽  
Duration Of Symptoms ◽  
Non Invasive ◽  
Viral Transport Medium ◽  
One Step ◽  
Antigen Tests ◽  
Low Sensitivity ◽  
Antigen Testing

AbstractObjectivesSaliva sampling could serve as an alternative non-invasive sample for SARS-CoV-2 diagnosis while rapid antigen testing (RAT) might help to mitigate the shortage of reagents sporadically encountered with RT-PCR. Thus, in the RESTART study we compared antigen and RT-PCR testing methods on nasopharyngeal (NP) swabs and salivary samples.MethodsWe conducted a prospective observational study among COVID-19 hospitalized patients between 10th December 2020 and 1st February 2021. Paired saliva and NP samples were investigated by RT-PCR (Cobas 6800, Roche-Switzerland) and by two rapid antigen tests: One Step Immunoassay Exdia® COVID-19 Ag (Precision Biosensor, Korea) and Standard Q® COVID-19 Rapid Antigen Test (Roche-Switzerland).ResultsA total of 58 paired NP-saliva specimens were collected. Thirty-two of 58 (55%) patients were hospitalized in the intensive care unit and the median duration of symptoms was 11 days (IQR 5-19). NP and salivary RT-PCR exhibited sensitivity of 98% and 69% respectively whereas the specificity of these RT-PCRs assays were of 100%. NP RAT exhibited much lower diagnostic performances with sensitivities of 35% and 41% for the Standard Q® and Exdia® assays respectively, when a wet-swab approach was used (i.e. when the swab was diluted in the viral transport medium (VTM) before testing). The sensitivity of the dry-swab approach was slightly better (47%). These antigen tests exhibited very low sensitivity (4 and 8%) when applied to salivary swabs.ConclusionsNasopharyngeal RT-PCR is the most accurate test for COVID-19 diagnosis in hospitalized patients. RT-PCR on salivary samples may be used when nasopharyngeal swabs are contraindicated. RAT are not appropriate for hospitalized patients.

scholarly journals Comparison Between a Standard and SalivaDirect RNA Extraction Protocol for Molecular Diagnosis of SARS-CoV-2 Using Nasopharyngeal Swab and Saliva Clinical Samples

2021 ◽  
Vol 9 ◽  
Author(s):  
Sofía N. Rodríguez Flores ◽  
Luis Mario Rodríguez-Martínez ◽  
Bernardita L. Reyes-Berrones ◽  
Nadia A. Fernández-Santos ◽  
Elthon J. Sierra-Moncada ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Sample Collection ◽  
Extraction Protocol ◽  
Assay Performance ◽  
Two Samples ◽  
Oropharyngeal Swab

During the COVID-19 pandemic, a certified laboratory of Tamaulipas, Mexico has processed over 100,000 samples of COVID-19 suspected patients, working a minimum of 100 tests daily. Thus, it would be beneficial for such certified laboratories nationwide to reduce the time and cost involved in performing the diagnosis of COVID-19, from sample collection, transportation to local lab, processing of samples, and data acquisition. Here, 30 nasopharyngeal swab and saliva samples from the same COVID-19 individuals were assessed by a standard nucleic acid extraction protocol, including protein lysis with proteinase K followed by binding to column, washing, and elution, and by the SalivaDirect protocol based on protein lysis, skipping the other steps to reduce processing time and costs. The genomic RNA was amplified using a SARS-CoV-2 Real-Time PCR kit. A variation (P > 0.05) in the 95% CIs = 72.6%–96.7% was noted by using the SalivaDirect protocol and saliva samples (sensitivity of 88.2%) in comparison to those of standard protocol with oropharyngeal swab samples (95% CIs = 97.5%–100%; sensitivity of 100%) as reported elsewhere. However, when using nasopharyngeal swab samples in the SalivaDirect protocol (sensitivity of 93.6%; 95% CIs = 79.2%–99.2%), it was in concordance (P < 0.05) with those of the standard one. The logical explanation to this was that two samples with Ct values of 38, and 40 cycles for gene E produced two false negatives in the SalivaDirect protocol in relation to the standard one; thus, there was a reduction of the sensitivity of 6.4% in the overall assay performance.

scholarly journals A faster and less costly alternative for RNA extraction of SARS-CoV-2 using proteinase k treatment followed by thermal shock

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248885
Author(s):  
Adolfo Marcelo Ñique ◽  
Fiorella Coronado-Marquina ◽  
Jairo Andrés Mendez Rico ◽  
María Paquita García Mendoza ◽  
Nancy Rojas-Serrano ◽  
...  
Keyword(s):  
Thermal Shock ◽  
Real Time ◽  
Internal Control ◽  
Rna Extraction ◽  
Cost Effective ◽  
Proteinase K ◽  
Rt Pcr ◽  
Critical Material ◽  
Proteinase K Treatment ◽  
Commercial Kit

One of the biggest challenges during the pandemic has been obtaining and maintaining critical material to conduct the increasing demand for molecular tests. Sometimes, the lack of suppliers and the global shortage of these reagents, a consequence of the high demand, make it difficult to detect and diagnose patients with suspected SARS-CoV-2 infection, negatively impacting the control of virus spread. Many alternatives have enabled the continuous processing of samples and have presented a decrease in time and cost. These measures thus allow broad testing of the population and should be ideal for controlling the disease. In this sense, we compared the SARS-CoV-2 molecular detection effectiveness by Real time RT-PCR using two different protocols for RNA extraction. The experiments were conducted in the National Institute of Health (INS) from Peru. We compared Ct values average (experimental triplicate) results from two different targets, a viral and internal control. All samples were extracted in parallel using a commercial kit and our alternative protocol–samples submitted to proteinase K treatment (3 μg/μL, 56°C for 10 minutes) followed by thermal shock (98°C for 5 minutes followed by 4°C for 2 minutes); the agreement between results was 100% in the samples tested. In addition, we compared the COVID-19 positivity between six epidemiological weeks: the initial two in that the Real time RT-PCR reactions were conducted using RNA extracted by commercial kit, followed by two other using RNA obtained by our kit-free method, and the last two using kit once again; they did not differ significantly. We concluded that our in-house method is an easy, fast, and cost-effective alternative method for extracting RNA and conducing molecular diagnosis of COVID-19.

scholarly journals Method versatility in RNA extraction-free PCR detection of SARS-CoV-2 in saliva samples

2021 ◽  
Author(s):  
Orchid M Allicock ◽  
Devyn Yolda-Carr ◽  
Rebecca Earnest ◽  
Mallery Breban ◽  
Noel Vega ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Rna Extraction ◽  
Health And Safety ◽  
Virus Detection ◽  
Cost Effective ◽  
Proteinase K ◽  
Sample Treatment ◽  
Simplified Approach ◽  
Safety Requirements ◽  
Qpcr Analysis

Early in the pandemic, a simple, open-source, RNA extraction-free RT-qPCR protocol for SARS-CoV-2 detection in saliva was developed and made widely available. This simplified approach (SalivaDirect) requires only sample treatment with proteinase K prior to PCR testing. However, feedback from clinical laboratories highlighted a need for a flexible workflow that can be seamlessly integrated into their current health and safety requirements for the receiving and handling of potentially infectious samples. To address these varying needs, we explored additional pre-PCR workflows. We built upon the original SalivaDirect workflow to include an initial incubation step (95°C for 30 minutes, 95°C for 5 minutes or 65°C for 15 minutes) with or without addition of proteinase K. The limit of detection for the workflows tested did not significantly differ from that of the original SalivaDirect workflow. When tested on de-identified saliva samples from confirmed COVID-19 individuals, these workflows also produced comparable virus detection and assay sensitivities, as determined by RT-qPCR analysis. Exclusion of proteinase K did not negatively affect the sensitivity of the assay. The addition of multiple heat pretreatment options to the SalivaDirect protocol increases the accessibility of this cost-effective SARS-CoV-2 test as it gives diagnostic laboratories the flexibility to implement the workflow which best suits their safety protocols.

scholarly journals 1208. Omadacycline In Vitro Activity Against Bacillus Anthracis

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S694-S695
Author(s):  
Alisa W Serio ◽  
H Carl Gelhaus ◽  
Noah Eichelberger ◽  
Henry S Heine ◽  
Diane M Anastasiou ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Resistant Strain ◽  
Clinical Laboratory ◽  
Independent Study ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Study Sites ◽  
Mic Value

Abstract Background Bacillus anthracis, the etiological agent of anthrax, is one of the agents most likely to be used in a biologic attack. Omadacycline previously has demonstrated potent in vitro and in vivo activity against B. anthracis. This project evaluated the in vitro activity of omadacycline against a larger set of B. anthracis strains across two laboratories. Methods Methods: Antibiotic susceptibility testing followed Clinical Laboratory Standard Institute methods against a collection of 53 B. anthracis strains at the University of Florida (UF) and 50 B. anthracis strains at MRIGlobal, representing human and animal isolates from North America, Africa, Europe, Asia, and Australia. Minimum inhibitory concentrations (MICs) for omadacycline and comparators at both sites (doxycycline, ciprofloxacin, levofloxacin, moxifloxacin) were determined by broth microdilution. Results Results: In the UF study, omadacycline demonstrated an MIC50 of 0.015 mg/L and an MIC90 of 0.03 mg/L against B. anthracis. Omadacycline MIC values were equal to or lower than doxycycline. In the MRIGlobal study, omadacycline demonstrated an MIC50 of 0.06 mg/L and an MIC90 of 0.06 mg/L (Table 1). All comparator MIC values were within ranges previously observed against these strains. Against a ciprofloxacin-resistant strain (MIC = 2 mg/L), omadacycline had an MIC value of 0.015 mg/L; against a doxycycline-resistant strain (MIC = 4 mg/L), omadacycline had an MIC value of 0.06 mg/L. Reproducibility was observed between the 2 laboratories for omadacycline in vitro activity against B. anthracis (Table 2). Table 1. MIC Concentration Summary for Omadacycline and Comparators Against B. anthracis Strains Table 2. Reproducibility of Omadacycline in Vitro Activity Against B. anthracis Strains Conclusion Based on the in vitro activity in both studies, omadacycline has the potential to be effective in treating anthrax infection. Reproducibility of omadacycline in vitro activity against B. anthracis was observed at 2 independent study sites. Disclosures Alisa W. Serio, PhD, Paratek Pharmaceuticals, Inc. (Employee, Shareholder) Diane M. Anastasiou, BA, Paratek Pharmaceuticals, Inc. (Consultant)

scholarly journals Studi Awal Analisis Molekuler Human Papillomavirus dari Apusan Glans dan Batang Penis

2021 ◽  
Vol 9 (4) ◽  
pp. 433
Author(s):  
Maya Savira ◽  
Resty Yuwandari ◽  
Yossi Maryanti ◽  
Rahmat Azhari Kemal ◽  
Donel S
Keyword(s):  
Human Papillomavirus ◽  
Rna Extraction ◽  
Viral Rna ◽  
Transport Medium ◽  
Viral Transport ◽  
Viral Transport Medium

Pria juga dapat mengalami keganasan akibat infeksi Human Papillomavirus (HPV) serta bertindak sebagai reservoir virus. Metode skrining HPV pada wanita telah terstandardisasi, namun belum ada standar metode skrining pada pria di Indonesia. Beberapa studi pada populasi pria di luar negeri menunjukkan potensi sampling pada daerah genitalia eksterna untuk skrining HPV. Tujuan: mengoptimasi metode skrining HPV secara molekuler pada pria. Metode: Responden adalah partner seksual wanita pansien kanker serviks di RSUD Arifin Achmad Provinsi Riau. Apusan dari glans dan batang penis diambil menggunakan nylon-flocked swab yang kemudian dimasukkan ke dalam 350µl viral transport medium terpisah. DNA diisolasi dari sampel yang kemudian dianalisis untuk mendeteksi gen human β-globin dan HPV. Hasil: Optimasi awal menunjukkan gen β-globin dapat terdeteksi dari hasil ekstraksi dengan kit Zeesan Viral RNA Extraction. Pita HPV hasil PCR dengan primer MY09 dan MY11 dapat muncul namun masih tipis. Simpulan: Studi awal ini menunjukkan bahwa apusan glans dan batang penis dapat digunakan untuk deteksi HPV secara molekuler pada pria, namun proses pengambilan sampel, ekstraksi DNA, dan PCR masih perlu dioptimasi.Kata kunci: apusan, glans, HPV, penis

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