Accessible LAMP-Enabled Rapid Test (ALERT) for detecting SARS-CoV-2

AbstractThe COVID-19 pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. PCR-based diagnostic tests are expensive, reliant on expensive centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests, that bind and detect the surface proteins of a virus, are rapid and inexpensive but suffer from high false negative rates. To address this problem, we have created an inexpensive, simple, and robust 60-minute Do-It-Yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/µl) and specificity (>97% True Negative Rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP).Our workflow, ALERT (Accessible LAMP-Enabled Rapid Test), incorporates the following features: 1) Increased shelf-life and ambient temperature storage by using wax layers to isolate enzymes from reaction, 2) Improved specificity by using sequence-specific QUASR reporters, 3) Increased sensitivity through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal, 4) Quality control with a nasopharyngeal-specific mRNA target, and 5) Co-detection of other respiratory viruses, such as Influenza B, by duplexing QUASR-modified RT-LAMP primer sets.The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for centralized labs and hospitals. With minimal effort, SARS-CoV-2-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms or nucleic acid-based markers.

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Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2

Viruses ◽

10.3390/v13050742 ◽

2021 ◽

Vol 13 (5) ◽

pp. 742

Author(s):

Ali Bektaş ◽

Michael F. Covington ◽

Guy Aidelberg ◽

Anibal Arce ◽

Tamara Matute ◽

...

Keyword(s):

Large Scale ◽

Point Of Care ◽

False Negative ◽

Rapid Test ◽

High Sensitivity ◽

Surface Proteins ◽

Influenza B ◽

True Negative ◽

Free Concentration ◽

Primer Sets

The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests that bind and detect the surface proteins of a virus are rapid and scalable but suffer from high false negative rates. To address this problem, an inexpensive, simple, and robust 60-minute do-it-yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/μL) and specificity (>97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents; (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters); (3) increased sensitivity, compared to methods without purification through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal; (4) quality control with a nasopharyngeal-specific mRNA target; and (5) co-detection of other respiratory viruses, such as influenza B, by multiplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms, or nucleic acid-based markers.

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Testing Denmark: A Danish nationwide surveillance study of COVID-19

10.1101/2021.08.10.21261777 ◽

2021 ◽

Author(s):

Kamille Fogh ◽

Jarl E Strange ◽

Bibi FSS Scharff ◽

Alexandra RR Eriksen ◽

Rasmus B Hasselbalch ◽

...

Keyword(s):

Large Scale ◽

Point Of Care ◽

Rapid Test ◽

Physical Contact ◽

Surveillance Study ◽

Epidemiological Surveillance ◽

Danish Population ◽

Positive Polymerase Chain Reaction ◽

Geographic Population ◽

Associated Risk Factors

Background National data on the spread of SARS-CoV-2 infection and knowledge on associated risk factors are important for understanding the course of the pandemic. Testing Denmark is a national large-scale epidemiological surveillance study of SARS-CoV-2 in the Danish population. Methods Between September and October 2020, approximately 1.3 million of 5.8 million Danish citizens (age > 15 years) were randomly invited to fill in an electronic questionnaire covering COVID-19 exposures and symptoms. The prevalence of SARS-CoV-2 antibodies was determined by Point-of Care rapid Test (POCT) distributed to participants home addresses. Findings In total 318,552 participants (24.5% invitees) completed the questionnaire and provided the result of the POCT. Of these, 2,519 (0.79%) were seropositive (median age 55 years) and women were more often seropositive than men, interquartile range (IQR) 42-64, 40.2% males. Of participants with a prior positive Polymerase Chain Reaction (PCR) test (n=1,828), 29.1% were seropositive in the POCT. Seropositivity increased with age irrespective of sex. Elderly participants (>61 years) reported less symptoms and had less frequently been tested for SARS-CoV-2 compared to younger participants. Seropositivity was associated with physical contact with SARS-CoV-2 infected individuals (Risk ratio (RR) 7.43, 95% CI: 6.57-8.41) and in particular household members (RR 17.70, 95% CI: 15.60-20.10). Home care workers had a higher risk of seropositivity (RR 2.09 (95% CI: 1.58-2.78) as compared to office workers. Geographic population density was not associated to seropositivity. A high degree of compliance with national preventive recommendations was reported (e.g., > 80% use of face masks), but no difference was found between seropositive and seronegative participants. Interpretation This study provides insight into the immunity of the Danish population seven to eight months after the first COVID-19 case in Denmark. The seroprevalence was lower than expected probably due to a low sensitivity of the POCT used or due to challenges relating to the reading of test results. Occupation or exposure in local communities were major routes of infection. As elderly participants were more often seropositive despite fewer symptoms and less PCR tests performed, more emphasis should be placed on testing this age group.

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Colorimetric Nanoplasmonics to Spot Hyperglycemia From Saliva

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2020.601216 ◽

2020 ◽

Vol 8 ◽

Author(s):

Paolo Donati ◽

Tania Pomili ◽

Luca Boselli ◽

Pier P. Pompa

Keyword(s):

Large Scale ◽

Point Of Care ◽

High Sensitivity ◽

Diabetic Patients ◽

Visual Response ◽

Colorimetric Sensing ◽

Chronic Patients ◽

Self Monitoring ◽

Sensing Platform ◽

Signal Color

Early diagnostics and point-of-care (POC) devices can save people’s lives or drastically improve their quality. In particular, millions of diabetic patients worldwide benefit from POC devices for frequent self-monitoring of blood glucose. Yet, this still involves invasive sampling processes, which are quite discomforting for frequent measurements, or implantable devices dedicated to selected chronic patients, thus precluding large-scale monitoring of the globally increasing diabetic disorders. Here, we report a non-invasive colorimetric sensing platform to identify hyperglycemia from saliva. We designed plasmonic multibranched gold nanostructures, able to rapidly change their shape and color (naked-eye detection) in the presence of hyperglycemic conditions. This “reshaping approach” provides a fast visual response and high sensitivity, overcoming common detection issues related to signal (color intensity) losses and bio-matrix interferences. Notably, optimal performances of the assay were achieved in real biological samples, where the biomolecular environment was found to play a key role. Finally, we developed a dipstick prototype as a rapid home-testing kit.

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Performance of SARS-CoV-2 Serology tests: Are they good enough?

10.1101/2020.11.13.20229625 ◽

2020 ◽

Author(s):

Isabelle Piec ◽

Emma English ◽

M Annette Thomas ◽

Samir Dervisevic ◽

William D Fraser ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Respiratory Infections ◽

Point Of Care ◽

False Negative ◽

Rapid Test ◽

Diagnostic Sensitivity ◽

Medical Community ◽

Antibody Detection ◽

Serological Tests ◽

Negative Results

AbstractBackgroundIn the emergency of the SARS-CoV-2 pandemic, great efforts were made to quickly provide serology testing to the medical community however, these methods have been introduced into clinical practice without the complete validation usually required by the regulatory organizations.MethodsSARS-CoV-2 patient samples (n=43) were analysed alongside pre-pandemic control specimen (n=50), confirmed respiratory infections (n=50), inflammatory polyarthritis (n=22) and positive for thyroid stimulating immunoglobulin (n=30). Imprecision, diagnostic sensitivity and specificity and concordance were evaluated on IgG serologic assays from EuroImmun, Epitope Diagnostics (EDI), Abbott Diagnostics and DiaSorin and a rapid IgG/IgM test from Healgen.ResultsEDI and EuroImmun imprecision was 0.02-14.0% CV. Abbott and DiaSorin imprecision (CV) ranged from 5.2% - 8.1% and 8.2% - 9.6% respectively. Diagnostic sensitivity of the assays were 100% (CI: 80-100%) for Abbott, EDI and EuroImmun and 95% (CI: 73-100%) for DiaSorin at ≥14 days post PCR. Only the Abbott assay had a diagnostic specificity of 100% (CI: 91-100%). EuroImmun cross-reacted in 3 non-SARS-CoV-2 respiratory infections and 2 controls. The DiaSorin displayed more false negative results and cross-reacted in six cases across all conditions tested. EDI had one cross-reactive sample. The Healgen rapid test showed excellent sensitivity and specificity. Overall, concordance of the assays ranged from 76.1% to 97.9%.ConclusionsSerological tests for SARS-CoV-2 showed good analytical performance. The head-to-head analysis of samples revealed differences in results that may be linked to the use of nucleocapsid or spike proteins. The point of care device tested demonstrated adequate performance for antibody detection.

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Methylene Blue as a Diagnosis and Screening Tool for Oral Cancer and Precancer

Otolaryngology ◽

10.1177/0194599820947686 ◽

2020 ◽

pp. 019459982094768

Author(s):

Se Hwan Hwang ◽

Sung Won Kim ◽

Eun A. Song ◽

Junuk Lee ◽

Do Hyun Kim

Keyword(s):

Methylene Blue ◽

Oral Cancer ◽

Diagnostic Accuracy ◽

False Positive ◽

Characteristic Curve ◽

False Positive Rate ◽

False Negative ◽

High Sensitivity ◽

Summary Receiver Operating Characteristic ◽

True Negative

Objectives To evaluate the accuracy of methylene blue (MB) for diagnosing oral cancer and precancer. Data Sources PubMed, Cochrane Database, Embase, Web of Science, SCOPUS, and Google Scholar. Review Methods Two authors working independently reviewed 6 databases from their dates of inception until April 2020. Studies exploring oral mucosal disorders as detected by MB were assessed. True-positive, true-negative, false-positive, and false-negative data were extracted for each study. Methodological quality was evaluated with the Quality Assessment of Diagnostic Accuracy Studies tool (v 2). Results Seven prospective and retrospective studies (N = 493) were included. The diagnostic odds ratio of MB was 20.017 (95% CI, 10.65-37.63, I2 = 23%). The area under the summary receiver operating characteristic curve was 0.699. Sensitivity was 0.903 (95% CI, 0.84-0.94, I2 = 54%), and specificity was 0.68 (95% CI, 0.60-0.75, I2 = 0%). The correlation between the sensitivity and the false-positive rate was –0.17, indicating an absence of heterogeneity. Conclusions Regarding diagnostic accuracy, MB had high sensitivity but low specificity, suggesting that it cannot be recommended as a replacement for the currently used standard of a scalpel biopsy with histologic assessment. Instead, it should be used as an adjunct to conventional assessment because of its low toxicity and price.

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Nasopharyngeal Panbio COVID-19 antigen performed at point-of-care has a high sensitivity in symptomatic and asymptomatic patients with higher risk for transmission and older age

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab059 ◽

2021 ◽

Author(s):

Mar Masiá ◽

Marta Fernández-González ◽

Manuel Sánchez ◽

Mar Carvajal ◽

José Alberto García ◽

...

Keyword(s):

Test Performance ◽

Point Of Care ◽

Rapid Test ◽

High Sensitivity ◽

Older Age ◽

Antigen Test ◽

Rt Pcr ◽

Asymptomatic Patients ◽

Clinical Scenarios ◽

Nasal Swabs

Abstract Background Performance of point-of-care tests in different clinical scenarios and on different samples remains undetermined. We comprehensively evaluated the performance of the nasopharyngeal Panbio COVID-19 antigen Rapid-Test-Device. Method Prospective study including consecutive patients attending three primary care centers (PCC) and an emergency department. The antigen test was performed at point-of-care in nasopharyngeal and nasal swabs, and in saliva. Positive and negative percent agreement (PPA, NPA) were calculated with the RT-PCR assay as reference standard. Results Of 913 patients included, 296 (32.3%) were asymptomatic and 690 (75.6%) came from the PCC. Nasopharyngeal swabs were collected from 913, nasal swabs from 659, and saliva from 611 patients. RT-PCR was positive in 196 (21.5%) nasopharyngeal samples (NPS). Overall PPA (95% CI) in NPS was 60.5% (53.3-67.4), and it was lower in nasal swabs (44.7%) and saliva (23.1%). Test performance in NPS was largely dependent on the cycle threshold (Ct) in RT-PCR, with PPA of 94% for Ct≤25 and 80% for Ct<30. In symptomatic patients, the PPA was 95% for Ct≤25; 85% for Ct<30, and 89% for the symptom triad of fever, cough and malaise. Performance was also dependent on age, with PPA of 100% in symptomatic patients >50 years with Ct<25. In asymptomatic patients, the PPA was 86% for Ct<25. In all cases, NPA was 100%. Conclusion The nasopharyngeal Panbio COVID-19 antigen test performed at point-of-care has a good sensitivity in symptomatic patients with Ct<30 and older age. The test was useful to identify asymptomatic patients with lower Ct values.

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Time scale performance of rapid antigen testing for SARS-COV-2: evaluation of ten rapid antigen assays

10.1101/2021.05.27.21257868 ◽

2021 ◽

Author(s):

Zakarya Abusrewil ◽

Inas M Alhudiri ◽

Hamza Kaal ◽

Salah Edin El Meshri ◽

Fawzi O Ebrahim ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Symptom Onset ◽

False Negative ◽

Rapid Test ◽

High Sensitivity ◽

Molecular Diagnostic ◽

Nasopharyngeal Swab ◽

Viral Loads ◽

Biotechnology Research ◽

Antigen Tests

Background: There is a great demand for more rapid tests for SARS-COV-2 detection to reduce waiting time, boost public health strategies for combating disease, decrease costs, and prevent overwhelming laboratory capacities. This study was conducted to assess the performance of 10 antigen-based rapid assays for the detection of SARS-CoV-2 in nasopharyngeal swab specimens. Methods: We analyzed 231 nasopharyngeal samples collected from October 2020-December 2020, from suspected COVID-19 cases and contacts of positive cases at Biotechnology Research Center laboratories, Tripoli, Libya. The performance of 10 COVID-19 Ag rapid test devices (Fluorecare, ESPLINE, RapiGen, Abbott Panbio, Flowflex, Acon, Assut Europe, Orient Gene, CerTest, Bioperfectus, AMP) for the detection of SARS-CoV-2 antigen was compared to RT-qPCR. Results: Among the 108 positive samples detected by RT-qPCR, the COVID-19 antigen (Ag) tests detected 83, giving a sensitivity of 76.85% (95% CI, 67.75- 84.43). 161 patients were symptomatic. The median cycle threshold was 25. The mean duration from symptom onset was 6.6 plus/minus 4.3 days. Sensitivity and specificity during the first 6 days of symptoms and in samples with high viral loads ct<25, was 96.4%. No false positives were detected by any of the Ag tests utilized in this study. False negative samples had a median Ct of 34 and average duration of onset of symptoms of 11.3 days (range=5-20). Conclusions: Rapid antigen test diagnosis has high sensitivity and specificity in early disease when patients present less than 7 days of symptom onset. Patients are encouraged to test as soon as they get COVID-19 related symptoms within 1 week and to seek medical advice within 24 hrs. if they develop disturbed smell/taste. The use of rapid antigen tests is important for controlling COVID-19 pandemic and reducing burden on molecular diagnostic laboratories.

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Shear Wave Elastography May Add a New Dimension to Ultrasound Evaluation of Thyroid Nodules: Case Series with Comparative Evaluation

Journal of Thyroid Research ◽

10.1155/2012/657147 ◽

2012 ◽

Vol 2012 ◽

pp. 1-6 ◽

Cited By ~ 25

Author(s):

Rafal Z. Slapa ◽

Antoni Piwowonski ◽

Wieslaw S. Jakubowski ◽

Jacek Bierca ◽

Kazimierz T. Szopinski ◽

...

Keyword(s):

Shear Wave ◽

Large Scale ◽

Thyroid Nodules ◽

False Negative ◽

Case Series ◽

Shear Wave Elastography ◽

True Negative ◽

Strain Elastography ◽

Negative Results ◽

Ultrasound Evaluation

Although elastography can enhance the differential diagnosis of thyroid nodules, its diagnostic performance is not ideal at present. Further improvements in the technique and creation of robust diagnostic criteria are necessary. The purpose of this study was to compare the usefulness of strain elastography and a new generation of elasticity imaging called supersonic shear wave elastography (SSWE) in differential evaluation of thyroid nodules. Six thyroid nodules in 4 patients were studied. SSWE yielded 1 true-positive and 5 true-negative results. Strain elastography yielded 5 false-positive results and 1 false-negative result. A novel finding appreciated with SSWE, were punctate foci of increased stiffness corresponding to microcalcifications in 4 nodules, some not visible on B-mode ultrasound, as opposed to soft, colloid-inspissated areas visible on B-mode ultrasound in 2 nodules. This preliminary paper indicates that SSWE may outperform strain elastography in differentiation of thyroid nodules with regard to their stiffness. SSWE showed the possibility of differentiation of high echogenic foci into microcalcifications and inspissated colloid, adding a new dimension to thyroid elastography. Further multicenter large-scale studies of thyroid nodules evaluating different elastographic methods are warranted.

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Use of Oral Fluid in a Rapid Syphilis Test Assay

Open Forum Infectious Diseases ◽

10.1093/ofid/ofx163.107 ◽

2017 ◽

Vol 4 (suppl_1) ◽

pp. S107-S108 ◽

Cited By ~ 1

Author(s):

Chelsea Shannon ◽

Claire Bristow ◽

Sasha Herbst De Cortina ◽

Jennifer Chang ◽

Jeffrey Klausner

Keyword(s):

Whole Blood ◽

Oral Fluid ◽

Point Of Care ◽

Fluid Collection ◽

Treponema Pallidum ◽

Rapid Test ◽

High Sensitivity ◽

The United States ◽

Collection Device ◽

Positive Results

Abstract Background From 2014 to 2015, the syphilis rate in the United States increased by 19%, reaching its highest rate since 1994. Currently, point-of-care syphilis assays use fingerstick or venipuncture whole blood to identify Treponema pallidum (TP) antibodies by qualitative immunoassay. However, patients and providers prefer oral fluid testing to whole blood testing. In this study, we aimed to determine whether a rapid syphilis test intended for use on whole blood could be used to detect TP antibodies in oral fluid. Methods Oral fluid was collected from 72 participants using the Super•SAL™ Oral Fluid Collection Device (Oasis Diagnostics®, Vancouver, WA). The device uses an absorbent cylindrical pad to collect and filter ~1 mlml of oral fluid. Oral fluid filtrate was tested using the SD Bioline Syphilis 3.0 rapid test (Alere Diagnostics, MA) following manufacturer directions for whole blood. TP particle agglutination (TPPA) and rapid plasma reagin (RPR) results derived from participants’ medical records were used as reference values. We used three different definitions as comparators: 1: TPPA reactive; 2: TPPA and RPR reactive and 3: TPPA reactive and RPR titer >1:4. Those with non-reactive TPPA and RPR results were considered seronegative. We calculated the sensitivity and specificity for definition 1 and sensitivity for definitions 2 and 3. We used the exact binomial method to determine 95% confidence intervals (CI). Results With definitions 1, 2, and 3, respectively, sensitivity was 83.3% (CI: 67.2, 93.6), 86.4% (CI: 65.1, 97.1), and 100% (CI: 71.5, 100). Specificity was 47.2% (CI: 36.5, 75.5). Conclusion The high sensitivity of the SD Bioline Syphilis 3.0 test using oral fluid suggests a strong potential for the development of accurate rapid oral syphilis tests. Sensitivity increased with higher RPR titer. False positive results may be due to the presence of non-venereal treponemal antibodies in oral fluid. Further research and development are needed to optimize specificity. Disclosures All authors: No reported disclosures.

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Sensitivity and Specificity of Gene Xpert in the Diagnosis of Spinal Tuberculosis: A Prospective Controlled Clinical Study

Global Spine Journal ◽

10.1177/2192568219858310 ◽

2019 ◽

Vol 10 (5) ◽

pp. 553-558

Author(s):

Anil M. Solanki ◽

Saumyajit Basu ◽

Amitava Biswas ◽

Subhendu Roy ◽

Aditya Banta

Keyword(s):

Positive Predictive Value ◽

Negative Predictive Value ◽

Sensitivity And Specificity ◽

Predictive Value ◽

Spinal Tuberculosis ◽

False Negative ◽

High Sensitivity ◽

Control Group ◽

Surgical Biopsy ◽

True Negative

Study Design: Prospective matched cohort study Objective: To evaluate the sensitivity and specificity of Gene Xpert in the diagnosis of spinal tuberculosis. Methods: From January 2016 to August 2018, Gene Xpert results were prospectively studied in 68 patients of clinicoradiologically suspected spinal tuberculosis (STB) and a control group (CG) of 92 patients, all of whom underwent computed tomography–guided/C-arm-guided/open surgical biopsy. Sensitivity, specificity, positive predictive value, and negative predictive value are obtained using standard equations. Results: Out of 68 cases of STB, Gene Xpert was positive in 62 (true positive: 62/68) and negative in 6 (false negative: 6/68). Gene Xpert was negative for all 92 cases of CG (true negative: 92/92, false positive 0/92). Thus, in our series, sensitivity of Gene Xpert is 91.18%, specificity is 100%, positive predictive value is 100%, and negative predictive value is 93.88%. Out of all cases of STB, 62/68 (91.18%) were Gene Xpert positive, but only 35/64 (54.69%) was acid-fast bacilli (AFB) culture positive and 53/60(88.33%) was histopathologically conclusive of TB. Also, Gene Xpert was positive in 7/7 (100%) cases of STB in which histopathology were inconclusive and 25/29 (86.21%) cases of STB in which AFB culture was negative. Conclusion: In STB, Gene Xpert clearly outperforms AFB culture and histopathology due to its high sensitivity and specificity apart from being rapid in diagnosis. Hence it is justified to diagnose spinal tuberculosis by Gene Xpert though histopathology is confirmative and AFB culture remains the gold standard.

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