Rapid, Sensitive, Label-Free Electrical Detection of SARS-CoV-2 in Nasal Swab Samples.

Rapid diagnosis of coronavirus disease 2019 (COVID-19) is key for the long-term control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) amid renewed threats of mutated SARS-CoV-2 around the world. Here, we report on an electrical label-free detection of SARS-CoV-2 in nasopharyngeal swab samples directly collected from outpatients or in saliva-relevant conditions by using a remote floating-gate field-effect transistor (RFGFET) with a 2-dimensional reduced graphene oxide (rGO) sensing membrane. RFGFET sensors demonstrate rapid detection (<5 min), a 90.6% accuracy from 8 nasal swab samples measured by 4 different devices for each sample, and a coefficient of variation (CV) < 6%. Also, RFGFET sensors display a limit of detection (LOD) of pseudo-SARS-CoV-2 that is 10 000-fold lower than enzyme-linked immunosorbent assays, with a comparable LOD to that of reverse transcription-polymerase chain reaction (RT-PCR) for patient samples. To achieve this, comprehensive systematic studies were performed regarding interactions between SARS-CoV-2 and spike proteins, neutralizing antibodies, and angiotensin-converting enzyme 2, as either a biomarker (detection target) or a sensing probe (receptor) functionalized on the rGO sensing membrane. Taken together, this work may have an immense effect on positioning FET bioelectronics for rapid SARS-CoV-2 diagnostics.

Remote Floating-Gate Field-Effect Transistor with 2-Dimensional Reduced Graphene Oxide Sensing Layer for Reliable Detection of SARS-CoV-2 Spike Proteins.

Despite intensive research of nanomaterials-based field-effect transistors (FETs) as a rapid diagnostic tool, it remains to be seen for FET sensors to be used for clinical applications due to a lack of stability, reliability, reproducibility, and scalability for mass production. Herein, we propose a remote floating-gate (RFG) FET configuration to eliminate device-to-device variations of two-dimensional reduced graphene oxide (rGO) sensing surfaces and most of the instability at the solution interface. Also, critical mechanistic factors behind the electrochemical instability of rGO such as severe drift and hysteresis were identified through extensive studies on rGO-solution interfaces varied by rGO thickness, coverage, and reduction temperature. rGO surfaces in our RFGFET structure displayed a Nernstian response of 54 mV/pH (from pH 2 to 11) with a 90% yield (9 samples out of total 10), coefficient of variation (CV) < 3%, and a low drift rate of 2%, all of which were calculated from the absolute measurement values. As proof-of-concept, we demonstrated highly reliable, reproducible, and label-free detection of spike proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a saliva-relevant media with concentrations ranging from 500 fg/mL to 5 µg/mL, with an R2 value of 0.984 and CV < 3%, and a guaranteed limit of detection at a few pg/mL. Taken together, this new platform may have an immense effect on positioning FET bioelectronics in a clinical setting for detecting SARS-CoV-2.

Sensitive detection and quantification of SARS-CoV-2 in saliva.

Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.

Implementation of a Sample Pooling Strategy for the Direct Detection of SARS-CoV-2 by Real-Time Polymerase Chain Reaction During the COVID-19 Pandemic.

OBJECTIVES: To report our institutional experience in devising and implementing a pooling protocol and process for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) testing over a 3-month period in the fall of 2020. METHODS: The widespread testing implemented in the United States for detecting SARS-CoV-2 infection in response to the coronavirus disease 2019 pandemic has led to a significant shortage of testing supplies and therefore has become a major impediment to the public health response. To date, several institutions have implemented sample pooling, but publications documenting these experiences are sparse. Nasal and nasopharyngeal samples collected from low-positivity (<5%) areas were tested in pools of five on the Roche cobas 6800 analyzer system. Routine SARS-CoV-2 RT-PCR turnaround times between sample collection to result reporting were monitored and compared before and after sample pooling implementation. RESULTS: A total of 4,131 sample pools were tested over a 3-month period (during which 39,770 RT-PCR results were reported from the Roche system), allowing our laboratory to save 13,824 tests, equivalent to a conservation rate of 35%. A 48-hour or less turnaround time was generally maintained throughout the pooling period. CONCLUSIONS: Sample pooling offers a viable means to mitigate shortfalls of PCR testing supplies in the ongoing pandemic without significantly compromising overall turnaround times.

Clinical Performance of the Point-of-Care cobas Liat for Detection of SARS-CoV-2 in 20 Minutes: a Multicenter Study.

Highly accurate testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the point of care (POC) is an unmet diagnostic need in emergency care and time-sensitive outpatient care settings. Reverse transcription-PCR (RT-PCR) technology is the gold standard for SARS-CoV-2 diagnostics. We performed a multisite U.S. study comparing the clinical performance of the first U.S. Food and Drug Administration (FDA)-authorized POC RT-PCR for detection of SARS-CoV-2 in 20 min, the cobas Liat SARS-CoV-2 and influenza A/B nucleic acid test, to the most widely used RT-PCR laboratory test, the cobas 68/8800 SARS-CoV-2 test. Clinical nasopharyngeal swab specimens from 444 patients with 357 evaluable specimens at five U.S. clinical laboratories were enrolled from 21 September 2020 to 23 October 2020. The overall agreement between the Liat and 68/8800 systems for SARS-CoV-2 diagnostics was 98.6% (352/357). Using Liat, positive percent agreement for SARS-CoV-2 was 100% (162/162) and the negative percent agreement was 97.4% (190/195). The Liat is an RT-PCR POC test that provides highly accurate SARS-CoV-2 results in 20 min with performance equivalent to that of high-throughput laboratory molecular testing. Rapid RT-PCR testing at the POC can enable more timely infection control and individual care decisions for coronavirus disease 2019.

Clinical Sensitivity of Severe Acute Respiratory Syndrome Coronavirus 2 Nucleic Acid Amplification Tests for Diagnosing Coronavirus Disease 2019.

Utilizing 34 348 severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) nucleic acid amplification test (NAAT) results from 2 health systems, we estimated the clinical sensitivity of a single SARS-CoV-2 NAAT. We found that SARS-CoV-2 NAAT has 82%-97% sensitivity for diagnosing coronavirus disease 2019 among symptomatic patients.

Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies.

As the Coronavirus 2019 (COVID-19) pandemic evolves, the development of immunoassays to help determine exposure and potentially predict immunity has become a pressing priority. In this report we present the performance of the EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for semi-quantitative detection of IgA and IgG antibodies in serum and plasma samples using recombinant S1 domain of the SARS-CoV-2 spike protein as antigen. Specimens from patients, with and without COVID-19 infection, were tested at the University of Chicago Clinical Microbiology and Immunology Laboratory. Of 86 samples from SARS-CoV-2 PCR-negative patients, including 28 samples positive for common human coronavirus strains, 76 tested negative and 10 tested positive for IgA (88.4% agreement, 95% CI: 79.9-93.6) while 84 tested negative and 2 tested positive for IgG (97.7% agreement, 95% CI: 91.9-99.6). Of 82 samples from SARS-CoV-2 PCR-positive patients, 14 tested negative and 68 tested positive for IgA (82.9% agreement, 95% CI: 73.4-89.5) while 27 tested negative and 55 tested positive for IgG (67.1% agreement, 95% CI: 56.3-76.3). Of samples collected ≥4 days after positive PCR, 38 of 42 (90.5% agreement, 95% CI: 77.9-96.2) were positive for IgA, and 42 of 42 (100% agreement, 95% CI: 91.6-100) were positive for IgG, respectively. The EUROIMMUN Anti-SARS-CoV-2 ELISA Assay demonstrated good sensitivity for detection of IgA and excellent sensitivity for detection of IgG antibodies from samples collected ≥4 days, after COVID-19 diagnosis by PCR. This assay demonstrated good specificity for IgA and excellent specificity for IgG and demonstrated only borderline cross reaction in 2 of the 28 samples from patients with common human coronaviruses infection, types NL63 and OC43.