scholarly journals CovidArray: A Microarray-Based Assay with High Sensitivity for the Detection of Sars-Cov-2 in Nasopharyngeal Swabs

Sensors ◽  
2021 ◽  
Vol 21 (7) ◽  
pp. 2490
Author(s):  
Francesco Damin ◽  
Silvia Galbiati ◽  
Stella Gagliardi ◽  
Cristina Cereda ◽  
Francesca Dragoni ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Solid Phase ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Great Sensitivity ◽  
Viral Rna ◽  
Analytical Sensitivity ◽  
Negative Results

A new coronavirus (SARS-CoV-2) caused the current coronavirus disease (Covid-19) epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions, RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false negatives are reported. Moreover, RT-qPCR requires up to 3–6 h with most of the time involved in RNA extraction from swab samples. We introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid-phase hybridization of fluorescently-labeled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 min. We correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity we were able to identify a false-negative sample. CovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows the reduction of both the amount of false-negative results and the total analysis time to about 2 h.

scholarly journals CovidArray: a microarray-based assay with high sensitivity for the detection of SARS-CoV-2 in nasopharyngeal swabs

2021 ◽  
Author(s):  
Francesco Damin ◽  
Silvia Galbiati ◽  
Stella Gagliardi ◽  
Cristina Cereda ◽  
Francesca Dragoni ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Solid Phase ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Digital Pcr ◽  
Great Sensitivity ◽  
Viral Rna ◽  
Analytical Sensitivity

AbstractBackgroundA new coronavirus (SARS-CoV-2) caused the current Covid-19 epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false-negative are reported. Moreover, RT-qPCR requires up to 3-6 hours with most of the time involved in RNA extraction from swab samples.MethodsWe introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid phase hybridization of fluorescently labelled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 minutes. To validate the CovidArray results, we exploited the high sensitivity of the droplet digital PCR (ddPCR) technique.ResultWe correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity that matches that of the ddPCR, we were able to identify a false-negative sample.ConclusionsCovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows to reduce both the amount of false negative results and the total analysis time to about 2 hours.

scholarly journals Patient DNA cross-reactivity of the CDC SARS-CoV-2 extraction control leads to an inherent potential for false negative results

2020 ◽  
Author(s):  
Adam P. Rosebrock
Keyword(s):  
Reverse Transcription ◽  
False Negative ◽  
Rna Extraction ◽  
Cross Reactivity ◽  
Sample Collection ◽  
Negative Results ◽  
False Negative Results ◽  
Important Diagnostic Tool ◽  
Control And Prevention ◽  
Careful Handling

AbstractTesting for RNA viruses such as SARS-CoV-2 requires careful handling of inherently labile RNA during sample collection, clinical processing, and molecular analysis. Tests must include fail-safe controls that affirmatively report the presence of intact RNA and demonstrate success of all steps of the assay. A result of “no virus signal” is insufficient for clinical interpretation: controls must also say “The reaction worked as intended and would have found virus if present.” Unfortunately, a widely used test specified by the US Centers for Disease Control and Prevention (CDC) incorporates a control that does not perform as intended and claimed. Detecting SARS-CoV-2 with this assay requires both intact RNA and successful reverse transcription. The CDC-specified control does not require either of these, due to its inability to differentiate human genomic DNA from reverse-transcribed RNA. Patient DNA is copurified from nasopharyngeal swabs during clinically-approved RNA extraction and is sufficient to return an “extraction control success” signal using the CDC design. As such, this assay fails-unsafe: truly positive patient samples return a false-negative result of “no virus detected, control succeeded” following any of several readily-encountered mishaps. This problem affects tens-of-millions of patients worth of shipped assays, but many of these flawed reagents have not yet been used. There is an opportunity to improve this important diagnostic tool. As demonstrated here, a re-designed transcript-specific control correctly monitors sample collection, extraction, reverse transcription, and qPCR detection. This approach can be rapidly implemented and will help reduce truly positive patients from being incorrectly given the all-clear.One Sentence SummaryA widely-used COVID-19 diagnostic is mis-designed and generates false-negative results, dangerously confusing “No” with “Don’t know” – but it’s fixable

scholarly journals Porcine reproductive and respiratory syndrome virus

2012 ◽  
Vol 24 (5) ◽  
pp. 855-866 ◽  
Author(s):  
Kerstin Wernike ◽  
Paolo Bonilauri ◽  
Malte Dauber ◽  
Jane Errington ◽  
Neil LeBlanc ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Respiratory Syndrome Virus ◽  
Analytical Sensitivity ◽  
Negative Results ◽  
False Negative Results ◽  
Ring Trial ◽  
Polymerase Chain ◽  
Commercial Kits ◽  
The Individual

To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays or commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed by sequencing and subsequent phylogenetic analysis. In summary, a major problem in PRRSV diagnostics by RT-qPCR is false-negative results. To achieve maximum safety in the molecular diagnosis of PRRSV, the combined usage of different assays or kits is highly recommended.

scholarly journals Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

scholarly journals Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6617
Author(s):  
Eva Rajh ◽  
Tina Šket ◽  
Arne Praznik ◽  
Petra Sušjan ◽  
Alenka Šmid ◽  
...  
Keyword(s):  
Oral Cavity ◽  
Reverse Transcription ◽  
Screening Program ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Diagnostic Sensitivity ◽  
Nucleic Acid Amplification ◽  
Nasopharyngeal Swab ◽  
Viral Spread ◽  
One Step

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

scholarly journals Visualization of Lesions of Metaphyses and Epiphyses of Bones in Newborns and Young Children

2021 ◽  
pp. 82-90
Author(s):  
N. A. Sholokhova
Keyword(s):  
Young Children ◽  
False Negative ◽  
Age Groups ◽  
High Sensitivity ◽  
Negative Results ◽  
False Negative Results ◽  
Inflammatory Processes ◽  
Group 5 ◽  
Magnetic Resonance Imaging Mri ◽  
Diagnostic Capabilities

The aim of this study was to determine the diagnostic capabilities of various methods of radiological diagnostics for lesions of the metaphyses and epiphyses of bones in newborns and young children.The study involved 108 children in the age group 5 days – 12 months with pathological changes in the pineal gland and bone metaphysis. The possibilities and advantages of standard radiography (СR), ultrasound examination (US) and magnetic resonance imaging (MRI) in the early and differential diagnosis of the osteomyelitis process and epiphyseolysis have been determined. High sensitivity (98 %), specificity (99 %) and accuracy (98 %) for ultrasound and sensitivity (94 %), specificity (89 %) and accuracy (95 %) of MRI in diagnosing osteomyelitis in patients of this age groups. At the same time, the possibilities of standard radiography at the stages of early diagnosis of inflammatory processes in the distal parts of the bones were limited due to a number of factors. The use of diagnostic algorithms greatly facilitates the work of a radiologist and reduces the number of false negative results during the initial treatment of patients.

CLINICAL APPLICATION OF COVID-19 REPORTING AND DATA SYSTEM IN COMPUTED TOMOGRAPHY OF BILATERAL PNEUMONIA DIAGNOSTIC

2021 ◽  
Vol 31 (4) ◽  
pp. 40-45
Author(s):  
Ugnė Kulnickaitė ◽  
Laura Dobrovaitė ◽  
Kamilė Grigaitė ◽  
Edvardas Jukna
Keyword(s):  
Computed Tomography ◽  
False Negative ◽  
High Sensitivity ◽  
Chest Ct ◽  
Data System ◽  
Negative Results ◽  
Radiological Society ◽  
Evaluation Scheme ◽  
False Negative Results ◽  
Standardized Reporting

Background: the 2019 coronavirus disease pandemic (COVID-19) has spread at an astonishing speed across the world, causing major morbidity and mortality. Computed tomography (CT) examination plays an important role in crisis areas in the diagnosis of COVID-19. COVID-19 Reporting and Data System (CO-RADS) has a five-point scale of suspicion for COVID-19 pneumonia in chest CT picture which standardizes the evaluation scheme and simplifies reporting. Aim: to summarise and present the role of COVID-19 Reporting and Data System in computed tomography of bilateral pneumonia diagnostic. Materials and methods: recently published studies were reviewed to evaluate COVID-19 Reporting and Data System scale as effective tool to detect COVID-19 pneumonia on chest CT scans. Databases from the subscription list of Lithuanian University of Health Sciences were selected: Medline (PubMed), SpringerLink and ScienceDirect. Results: chest CT features, as bilateral involvement, subpleural or peripherally distributed GGO, consolidation, reticulation, crazy paving pattern, air bronchogram signs, intralobular septal thickening, pulmonary vascular enlargement, are considered to be characteristic manifestations of COVID-19 infection. Studies show that Dutch Radiological Society presented CO-RADS scale sensitivity and specificity may vary from 61-88% and 66,4-98%, respectively. Conclusion: chest CT scan has a high sensitivity for COVID-19 diagnosis and could reduce false negative results obtained from RT-PCR tests. Furthermore, a standardized reporting system could increase clarification, minimize reporting variability and help radiologists recognize the results they observe, especially, for less experienced specialists.

Immunogenicity of Golimumab and its Clinical Relevance in Patients With Ulcerative Colitis

2019 ◽  
Vol 25 (9) ◽  
pp. 1532-1540 ◽  
Author(s):  
Omoniyi J Adedokun ◽  
George R Gunn ◽  
Jocelyn H Leu ◽  
Cynthia Gargano ◽  
Zhenhua Xu ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
False Negative ◽  
Injection Site Reaction ◽  
High Sensitivity ◽  
Fold Increase ◽  
Drug Interference ◽  
Negative Results ◽  
Drug Concentrations ◽  
Antidrug Antibody ◽  
Antibody Titers

Abstract Background Antidrug antibody (ADA) detection with standard bridging enzyme immunoassays (EIA) can yield false-negative results or underestimate titers through drug interference. A more sensitive assay was needed to determine clinical impact of antigolimumab antibodies. Methods A high-sensitivity, drug-tolerant EIA (DT-EIA) was developed and cross-validated against the original EIA, and samples from induction/maintenance studies in golimumab-treated patients with ulcerative colitis were analyzed for ADAs using both methods. Immunogenicity results were compared, and pharmacokinetic, efficacy, and safety associations were evaluated. Results An 8-fold increase in ADA-positive patients (21.8% DT-EIA vs 2.8% EIA) reflected DT-EIA improved sensitivity and drug tolerance. Most newly detected ADA-positive patients (using DT-EIA) had low antibody titers, whereas most with high antibody titers were ADA-positive with original EIA. With DT-EIA, week 44 median trough serum golimumab concentrations among ADA-positive patients were approximately half vs ADA-negative (0.51 vs 0.85 µg/mL [50 mg q4w]; 0.85 vs 1.60 µg/mL [100 mg q4w]). Antidrug antibody impact on golimumab concentrations was more notable at titers ≥1:100. During induction, ADAs had no notable impact on efficacy. During maintenance, proportions of patients maintaining clinical response through week 54 were lower using DT-EIA: 38.1% ADA-positive and 52.8% ADA-negative. Antidrug antibody status had no impact on injection-site reaction incidence. Conclusions A more sensitive DT-EIA identified higher proportions of ADA-positive patients. A trend of decreasing drug concentrations with increasing ADA titers was observed. Pharmacokinetic impact was better elucidated with DT-EIA. Although development of ADA did not preclude efficacy, a trend toward decreased efficacy in ADA-positive vs ADA-negative patients was observed during maintenance treatment. Antidrug antibody status did not impact safety.

scholarly journals Specific circulating immune complexes in acute chagas' disease

1987 ◽  
Vol 29 (1) ◽  
pp. 26-32 ◽  
Author(s):  
Ricardo Corral ◽  
Héctor Freilij ◽  
Saúl Grinstein
Keyword(s):  
Chagas Disease ◽  
Immune Complexes ◽  
False Negative ◽  
High Sensitivity ◽  
Circulating Immune Complexes ◽  
Chronic Patients ◽  
Negative Results ◽  
Acute Toxoplasmosis ◽  
Acute Chagas Disease ◽  
Cruzi Infection

The presence of circulating immune complexes formed by IgM and IgG (CIC-IgM and CIC-IgG) was investigated, using antigen-specific enzyme-immunoassays (ELISA), in 30 patients with acute Chagas' disease who showed parasitemia and inoculation chagoma. Control population consisted of patients with chronic T. cruzi infection (30), acute toxoplasmosis 10), leishmaniasis (8), rheumatoid arthritis (3) and healthy individuals with negative serology for Chagas* disease (30). Acute chagasic patients were 100% CIC-IgG and 96.66% CIC-IgM positive whereas immunofluorescence tests yielded 90% and 86.66% of positivity for specific IgG and IgM antibodies, respectively. Chronic patients were 68% CIC-IgG and 0% CIC-IgM positive. The 30 negative and the 21 cross-reaction controls proved negative for ELISA (CIC-IgM and CIC-IgG). The high sensitivity of ELISA assays would allow early immunologic diagnosis, as well as prompt treatment, of acute T. cruzi infection, thus eliminating the problem of the false-positive and false-negative results which affects traditional methods for detection of circulating antibodies.

scholarly journals Evaluation of COVID-19 RT-qPCR Test in Multi sample Pools

2020 ◽  
Vol 71 (16) ◽  
pp. 2073-2078 ◽  
Author(s):  
Idan Yelin ◽  
Noga Aharony ◽  
Einat Shaer Tamar ◽  
Amir Argoetti ◽  
Esther Messer ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
False Negative Rate ◽  
Positive Sample ◽  
Recent Emergence ◽  
Single Positive ◽  
Polymerase Chain ◽  
Pool Test ◽  
Current Screening

Abstract Background The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a current pandemic of unprecedented scale. Although diagnostic tests are fundamental to the ability to detect and respond, overwhelmed healthcare systems are already experiencing shortages of reagents associated with this test, calling for a lean immediately applicable protocol. Methods RNA extracts of positive samples were tested for the presence of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction, alone or in pools of different sizes (2-, 4-, 8-, 16-, 32-, and 64-sample pools) with negative samples. Transport media of additional 3 positive samples were also tested when mixed with transport media of negative samples in pools of 8. Results A single positive sample can be detected in pools of up to 32 samples, using the standard kits and protocols, with an estimated false negative rate of 10%. Detection of positive samples diluted in even up to 64 samples may also be attainable, although this may require additional amplification cycles. Single positive samples can be detected when pooling either after or prior to RNA extraction. Conclusions As it uses the standard protocols, reagents, and equipment, this pooling method can be applied immediately in current clinical testing laboratories. We hope that such implementation of a pool test for coronavirus disease 2019 would allow expanding current screening capacities, thereby enabling the expansion of detection in the community, as well as in close organic groups, such as hospital departments, army units, or factory shifts.

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