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2.
Clin Rev Allergy Immunol ; 59(1): 89-100, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-1139384

ABSTRACT

The COVID-19 pandemic is a significant global event in the history of infectious diseases. The SARS-CoV-2 appears to have originated from bats but is now easily transmissible among humans, primarily through droplet or direct contact. Clinical features of COVID-19 include high fever, cough, and fatigue which may progress to ARDS. Respiratory failure can occur rapidly after this. The primary laboratory findings include lymphopenia and eosinopenia. Elevated D-dimer, procalcitonin, and CRP levels may correlate with disease severity. Imaging findings include ground-glass opacities and patchy consolidation on CT scan. Mortality is higher in patients with hypertension, cardiac disease, diabetes mellitus, cancer, and COPD. Elderly patients are more susceptible to severe disease and death, while children seem to have lower rates of infection and lower mortality. Diagnostic criteria and the identification of persons under investigation have evolved as more data has emerged. However, the approach to diagnosis is still very variable from region to region, country to country, and even among different hospitals in the same city. The importance of a clinical pathway to implement the most effective and relevant diagnostic strategy is of critical importance to establish the control of this virus that is responsible for more and more deaths each day.


Subject(s)
Antibodies, Viral/immunology , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Lung/diagnostic imaging , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Algorithms , Betacoronavirus/immunology , Critical Pathways , Early Diagnosis , Evidence-Based Practice , False Negative Reactions , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Medical History Taking , Pandemics , Patient Isolation , Quarantine , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Serologic Tests/methods , Severity of Illness Index , Tomography, X-Ray Computed
3.
Diagn Microbiol Infect Dis ; 99(3): 115260, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1139480

ABSTRACT

The BioFire® COVID-19 Test and Respiratory Panel 2.1 (RP2.1) are rapid, fully automated assays for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs. In the case of the RP2.1, an additional 21 viral and bacterial pathogens can be detected. Both tests have received emergency use authorization from the U.S. Food & Drug Administration and Interim Order authorization from Health Canada for use in clinical laboratories. We evaluated the performance characteristics of these tests in comparison to a laboratory-developed real-time PCR assay targeting the viral RNA-dependent RNA polymerase and E genes. A total of 78 tests were performed using the BioFire COVID-19 Test, including 30 clinical specimens and 48 tests in a limit of detection study; 57 tests were performed using the RP2.1 for evaluation of SARS-CoV-2 detection, including 30 clinical specimens and 27 tests for limit of detection. Results showed 100% concordance between the BioFire assays and the laboratory-developed test for all clinical samples tested, and acceptable performance of both BioFire assays at their stated limits of detection. Conclusively, the BioFire COVID-19 Test and RP2.1 are highly sensitive assays that can be effectively used in the clinical laboratory for rapid SARS-CoV-2 testing.


Subject(s)
/methods , Nasopharynx/virology , /isolation & purification , /standards , Clinical Laboratory Techniques/methods , Diagnostic Tests, Routine , Humans , Limit of Detection , Multiplex Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction/methods , Reproducibility of Results , Sensitivity and Specificity
4.
Int J Mol Sci ; 22(5)2021 Feb 28.
Article in English | MEDLINE | ID: covidwho-1120492

ABSTRACT

Although molecular testing, and RT-qPCR in particular, has been an indispensable component in the scientific armoury targeting SARS-CoV-2, there are numerous falsehoods, misconceptions, assumptions and exaggerated expectations with regards to capability, performance and usefulness of the technology. It is essential that the true strengths and limitations, although publicised for at least twenty years, are restated in the context of the current COVID-19 epidemic. The main objective of this commentary is to address and help stop the unfounded and debilitating speculation surrounding its use.


Subject(s)
/methods , /virology , Molecular Diagnostic Techniques/methods , Real-Time Polymerase Chain Reaction/methods , /isolation & purification , Clinical Laboratory Techniques/methods , Humans , RNA, Viral/analysis , RNA, Viral/genetics , Sensitivity and Specificity
6.
PLoS One ; 16(3): e0248042, 2021.
Article in English | MEDLINE | ID: covidwho-1115310

ABSTRACT

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).


Subject(s)
/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , /genetics , /genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , DNA Primers/genetics , Humans , Nucleocapsid Proteins/genetics , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription/genetics , Sensitivity and Specificity
7.
Am J Clin Pathol ; 153(6): 715-718, 2020 05 05.
Article in English | MEDLINE | ID: covidwho-1109161

ABSTRACT

OBJECTIVES: To establish the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2. METHODS: The most efficient pool size was determined to be five specimens using a web-based application. From this analysis, 25 experimental pools were created using 50 µL from one SARS-CoV-2 positive nasopharyngeal specimen mixed with 4 negative patient specimens (50 µL each) for a total volume of 250 µL. Viral RNA was subsequently extracted from each pool and tested using the CDC SARS-CoV-2 RT-PCR assay. Positive pools were consequently split into individual specimens and tested by extraction and PCR. This method was also tested on an unselected group of 60 nasopharyngeal specimens grouped into 12 pools. RESULTS: All 25 pools were positive with cycle threshold (Ct) values within 0 and 5.03 Ct of the original individual specimens. The analysis of 60 specimens determined that 2 pools were positive followed by identification of 2 individual specimens among the 60 tested. This testing was accomplished while using 22 extractions/PCR tests, a savings of 38 reactions. CONCLUSIONS: When the incidence rate of SARS-CoV-2 infection is 10% or less, group testing will result in the saving of reagents and personnel time with an overall increase in testing capability of at least 69%.


Subject(s)
Clinical Laboratory Techniques/economics , Clinical Laboratory Techniques/methods , Medical Laboratory Personnel/economics , Specimen Handling/economics , Specimen Handling/methods , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/instrumentation , Clinical Laboratory Techniques/standards , Coronavirus Infections/diagnosis , Coronavirus Infections/economics , Humans , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/economics , Specimen Handling/standards
8.
PLoS Negl Trop Dis ; 15(3): e0009227, 2021 03.
Article in English | MEDLINE | ID: covidwho-1110082

ABSTRACT

Since its first emergence in 2012, cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) have continued to occur. At the end of January 2020, 2519 laboratory confirmed cases with a case-fatality rate of 34.3% have been reported. Approximately 84% of human cases have been reported in the tropical region of Saudi Arabia. The emergence of MERS-CoV has highlighted need for a rapid and accurate assay to triage patients with a suspected infection in a timely manner because of the lack of an approved vaccine or an effective treatment for MERS-CoV to prevent and control potential outbreaks. In this study, we present two rapid and visual nucleic acid assays that target the MERS-CoV UpE and N genes as a panel that combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). This test panel was designed to improve the diagnostic accuracy through dual-target screening after referencing laboratory testing guidance for MERS-CoV. The limit of detection was 1.2×101 copies/µl viral RNA for the UpE assay and 1.2 copies/µl viral RNA for the N assay, with almost consistent with the sensitivity of the RT-qPCR assays. The two assays exhibited no cross-reactivity with multiple CoVs, including the bat severe acute respiratory syndrome related coronavirus (SARSr-CoV), the bat coronavirus HKU4, and the human coronaviruses 229E, OC43, HKU1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the panel does not require sophisticated equipment and provides rapid detection within 30 min. This panel displays good sensitivity and specificity and may be useful to rapidly detect MERS-CoV early during an outbreak and for disease surveillance.


Subject(s)
Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , Middle East Respiratory Syndrome Coronavirus/genetics , Molecular Diagnostic Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription , Saudi Arabia/epidemiology , Sensitivity and Specificity , Viral Nonstructural Proteins/genetics
9.
PLoS One ; 16(2): e0247524, 2021.
Article in English | MEDLINE | ID: covidwho-1099931

ABSTRACT

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 has remained essential for tracking and containing the virus. SARS-CoV-2 testing capabilities are still limited in many countries. Here, we explore the use of conventional RNA purification as an alternative to automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical swab specimens were extracted by conventional phenol-chloroform RNA purification and compared to commercial platforms for RNA extraction and the fully integrated Cobas®6800 diagnostic system. Our results show that the conventional RNA extraction is fully comparable to modern automated systems regarding analytical sensitivity and specificity with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, the method is easily scalable and implemented in conventional laboratories as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2.


Subject(s)
/virology , RNA, Viral/isolation & purification , /genetics , /diagnosis , Chloroform/chemistry , Clinical Laboratory Techniques/methods , Humans , Molecular Diagnostic Techniques/methods , Pandemics , Phenol/chemistry , RNA/genetics , RNA/isolation & purification , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Specimen Handling/methods
10.
PLoS One ; 16(2): e0245914, 2021.
Article in English | MEDLINE | ID: covidwho-1088751

ABSTRACT

In 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. SARS-CoV-2 patient samples (n = 43) were analyzed 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. EDI 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 was 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%. Serological 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.


Subject(s)
/methods , /immunology , Antibodies, Viral/blood , Brazil/epidemiology , /immunology , /trends , Clinical Laboratory Techniques/methods , Cross Reactions , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Pandemics , Point-of-Care Systems , Sensitivity and Specificity
11.
J Virol Methods ; 291: 114102, 2021 05.
Article in English | MEDLINE | ID: covidwho-1085514

ABSTRACT

Multiple nucleic acid amplification tests (NATs) are available for the detection of SARS-CoV-2 in clinical specimens, including Laboratory Developed Tests (LDT), commercial high-throughput assays and point-of-care tests. Some assays were just recently released and there is limited data on their clinical performance. We compared the Xpert® Xpress SARS-CoV-2 (Cepheid) and Vivalytic VRI Panel (Schnelltest COVID-19) (Bosch) point-of-care tests with four high-throughput assays and one LDT, the cobas® SARS-CoV-2 test (Roche), the Allplex™ 2019-nCoV Assay (Seegene), the SARS-CoV-2 AMP (Abbott) Kit, the RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (altona) as well as an assay using a SARS-CoV-2 RdRP gene specific primer and probe set. Samples from patients with confirmed SARS-CoV-2 infection, samples from the first and second SARS-CoV-2-PCR External Quality Assessment (EQA) (INSTAND e.V.) and a 10-fold serial dilution of a SARS-CoV-2 cell culture (SARS-CoV-2 Frankfurt 1) supernatant were examined. We determined that the NAT assays examined had a high specificity. Assays using the N gene as target demonstrated the highest sensitivity in the serial dilution panel, while all examined NAT assays showed a comparable sensitivity when testing clinical and EQA samples.


Subject(s)
/methods , High-Throughput Screening Assays/methods , Point-of-Care Testing , /isolation & purification , /methods , Clinical Laboratory Techniques/methods , Humans , Nucleic Acid Amplification Techniques/methods , Sensitivity and Specificity
12.
JAMA Netw Open ; 4(2): e2037129, 2021 02 01.
Article in English | MEDLINE | ID: covidwho-1077483

ABSTRACT

Importance: The reopening of colleges and universities in the US during the coronavirus disease 2019 (COVID-19) pandemic is a significant public health challenge. The development of accessible and practical approaches for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in the college population is paramount for deploying recurrent surveillance testing as an essential strategy for virus detection, containment, and mitigation. Objective: To determine the prevalence of SARS-CoV-2 in asymptomatic participants in a university community by using CREST (Cas13-based, rugged, equitable, scalable testing), a CRISPR-based test developed for accessible and large-scale viral screening. Design, Setting, and Participants: For this cohort study, a total of 1808 asymptomatic participants were screened for SARS-CoV-2 using a CRISPR-based assay and a point-of-reference reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) test. Viral prevalence in self-collected oropharyngeal swab samples collected from May 28 to June 11, 2020, and from June 23 to July 2, 2020, was evaluated. Exposures: Testing for SARS-CoV-2. Main Outcomes and Measures: SARS-CoV-2 status, viral load, and demographic information of the study participants were collected. Results: Among the 1808 participants (mean [SD] age, 27.3 [11.0] years; 955 [52.8%] female), 732 underwent testing from May to early June (mean [SD] age, 28.4 [11.7] years; 392 [53.6%] female). All test results in this cohort were negative. In contrast, 1076 participants underwent testing from late June to early July (mean [SD] age, 26.6 [10.5] years; 563 [52.3%] female), with 9 positive results by RT-qPCR. Eight of these positive samples were detected by the CRISPR-based assay and confirmed by Clinical Laboratory Improvement Amendments-certified diagnostic testing. The mean (SD) age of the positive cases was 21.7 (3.3) years; all 8 individuals self-identified as students. These metrics showed that a CRISPR-based assay was effective at capturing positive SARS-CoV-2 cases in this student population. Notably, the viral loads detected in these asymptomatic cases resemble those seen in clinical samples, highlighting the potential of covert viral transmission. The shift in viral prevalence coincided with the relaxation of stay-at-home measures. Conclusions and Relevance: These findings reveal a shift in SARS-CoV-2 prevalence in a young and asymptomatic population and uncover the leading edge of a local outbreak that coincided with rising case counts in the surrounding county and the state of California. The concordance between CRISPR-based and RT-qPCR testing suggests that CRISPR-based assays are reliable and offer alternative options for surveillance testing and detection of SARS-CoV-2 outbreaks, as is required to resume operations in higher-education institutions in the US and abroad.


Subject(s)
/diagnosis , Clinical Laboratory Techniques/methods , Clustered Regularly Interspaced Short Palindromic Repeats , Mass Screening/methods , Reverse Transcriptase Polymerase Chain Reaction , Universities , Adolescent , Adult , Cohort Studies , Disease Outbreaks , Female , Humans , Male , Pandemics , RNA-Directed DNA Polymerase , Students , Viral Load , Young Adult
13.
J Virol Methods ; 291: 114086, 2021 05.
Article in English | MEDLINE | ID: covidwho-1071741

ABSTRACT

The worldwide demand for SARS-CoV-2 RT-PCR testing resulted in a shortage of diagnostic kits. RNA extraction step constitutes a major bottleneck to perform diagnostic. The aim of this study was to assess performances of different extraction-free SARS-CoV-2 RT-PCR assays compared to a reference RT-PCR assay. The panel of evaluation consisted of 94 samples: 69 positive and 25 negative for SARS-CoV-2 by reference RT-PCR. Three extraction-free RT-PCR assays were assessed: (i) PrimeDirect® Probe RT-qPCR Mix (Takara), (ii) PrimeScript®RT-PCR (Takara), and (iii) SARS-CoV-2 SANSURE®BIOTECH Novel Coronavirus (Sansure). The overall sensitivity of PrimeDirect, PrimeScript and Sansure assays was 55.1 %, 69.6 % and 69.6 %, respectively. The sensitivity increased among samples with Ct<30: 91.9 % (n = 34/37), 89.2 % (n = 33/37) and 94.6 % (n = 35/37) for PrimeDirect, PrimeScript and Sansure assays, respectively. The specificity was 88 %, 100 % and 100 % for PrimeDirect, PrimeScript and Sansure assays, respectively. In the present study, we showed a good sensitivity of extraction-free PCR assays, especially for high viral loads (Ct<30), except PrimeDirect that displayed imperfect sensitivity and specificity. Despite a lower sensitivity for low viral loads, extraction-free reagents can provide a valuable option, cheaper, easier and less reagent consuming for SARS-CoV-2 diagnostic, especially in laboratory with lower experience and equipment for molecular assays.


Subject(s)
/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , /isolation & purification , /virology , Clinical Laboratory Techniques/methods , RNA, Viral/genetics , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Viral Load
14.
Biotechniques ; 69(4): 317-325, 2020 10.
Article in English | MEDLINE | ID: covidwho-1067502

ABSTRACT

PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the technologies that have evolved from the original PCR method. Currently, there are two main areas of PCR utilization in bioscience: high-throughput PCR systems and microfluidics-based PCR devices for point-of-care (POC) applications. We also discuss the commercialization of these techniques and conclude with a look into their modifications and use in innovative areas of biomedicine. For example, real-time reverse transcription PCR is the gold standard for SARS-CoV-2 diagnoses. It could also be used for POC applications, being a key component of the sample-to-answer system.


Subject(s)
Polymerase Chain Reaction/methods , Animals , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/instrumentation , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Equipment Design , Humans , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Point-of-Care Systems , Polymerase Chain Reaction/instrumentation
15.
Life Sci ; 273: 119117, 2021 May 15.
Article in English | MEDLINE | ID: covidwho-1065431

ABSTRACT

Biosensors are important devices in clinical diagnostics, food processing, and environmental monitoring for detecting various analytes, especially viruses. These biosensors provide rapid and effective instruments for qualitative and quantitative detection of infectious diseases in real-time. Here, we report the development of biosensors based on various techniques. Additionally, we will explain the mechanisms, advantages, and disadvantages of the most common biosensors that are currently used for viral detection, which could be optical (e.g., surface-enhanced Raman scattering (SERS), Surface plasmon resonance (SPR)) and electrochemical biosensors. Based on that, this review recommends methods for efficient, simple, low-cost, and rapid detection of SARS-CoV-2 (the causative agent of COVID-19) that employ the two types of biosensors depending on attaching hemoglobin ß-chain and binding of specific antibodies with SARS-CoV-2 antigens, respectively.


Subject(s)
Biosensing Techniques/methods , /diagnosis , Biosensing Techniques/instrumentation , /instrumentation , Clinical Laboratory Techniques/instrumentation , Clinical Laboratory Techniques/methods , Equipment Design , Humans , /isolation & purification
16.
J Infect Dis ; 223(2): 206-213, 2021 02 03.
Article in English | MEDLINE | ID: covidwho-1060913

ABSTRACT

BACKGROUND: Recent advances in CRISPR-based diagnostics suggest that DETECTR, a combination of reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) and subsequent Cas12 bystander nuclease activation by amplicon-targeting ribonucleoprotein complexes, could be a faster and cheaper alternative to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) without sacrificing sensitivity and/or specificity. METHODS: In this study, we compare DETECTR with qRT-PCR to diagnose coronavirus disease 2019 on 378 patient samples. Patient sample dilution assays suggest a higher analytical sensitivity of DETECTR compared with qRT-PCR; however, this was not confirmed in this large patient cohort, where we report 95% reproducibility between the 2 tests. RESULTS: These data showed that both techniques are equally sensitive in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) providing additional value of DETECTR to the currently used qRT-PCR platforms. For DETECTR, different guide ribonucleic acids can be used simultaneously to obviate negative results due to mutations in N-gene. Lateral flow strips, suitable as a point-of-care test, showed a 100% correlation to the high-throughput DETECTR assay. More importantly, DETECTR was 100% specific for SARS-CoV-2 relative to other human coronaviruses. CONCLUSIONS: Because there is no need for specialized equipment, DETECTR could be rapidly implemented as a complementary technically independent approach to qRT-PCR thereby increasing the testing capacity of medical microbiological laboratories and relieving the existent PCR platforms for routine non-SARS-CoV-2 diagnostic testing.


Subject(s)
/methods , /virology , Reverse Transcriptase Polymerase Chain Reaction/methods , /isolation & purification , Clinical Laboratory Techniques/methods , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Point-of-Care Testing , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reference Standards , Reproducibility of Results , /genetics
17.
Obstet Gynecol ; 136(5): 1016-1020, 2020 11.
Article in English | MEDLINE | ID: covidwho-1059680

ABSTRACT

BACKGROUND: The risk of vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unknown. Positive reverse-transcription polymerase chain reaction (RT-PCR) test results for SARS-CoV-2 infection in neonates and placental tissue have been reported, and immunoglobulin M antibodies have been detected in neonates born to mothers with infection. CASES: The first case is a woman at 22 3/7 weeks of gestation with coronavirus disease 2019 (COVID-19) who was admitted to the intensive care unit. In the second case, the patient remained at home with mild symptoms, starting at 20 weeks of gestation. In both cases, fetal skin edema was observed on ultrasound examination while maternal SARS-COV-2 RT-PCR test results were positive and resolved when maternal SARS-COV-2 RT-PCR test results became negative. The RT-PCR test result for SARS-CoV-2 in amniotic fluid was negative in both cases. The two pregnancies are ongoing and uneventful. CONCLUSION: Transient fetal skin edema noted in these two patients with COVID-19 in the second trimester may represent results of fetal infection or altered fetal physiology due to maternal disease or may be unrelated to the maternal illness.


Subject(s)
Coronavirus Infections , Fetal Monitoring/methods , Hydrops Fetalis/diagnosis , Pandemics , Pneumonia, Viral , Pregnancy Complications, Infectious , Skin/diagnostic imaging , Adult , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/physiopathology , Coronavirus Infections/therapy , Female , Gestational Age , Humans , Immunoglobulin M/blood , Infant, Newborn , Infectious Disease Transmission, Vertical/prevention & control , Middle Aged , Placenta/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/physiopathology , Pneumonia, Viral/therapy , Pregnancy , Pregnancy Complications, Infectious/diagnosis , Pregnancy Complications, Infectious/physiopathology , Pregnancy Complications, Infectious/therapy , Pregnancy Complications, Infectious/virology , Respiration, Artificial/methods , Treatment Outcome , Ultrasonography, Prenatal/methods
18.
Sci Rep ; 11(1): 2402, 2021 01 28.
Article in English | MEDLINE | ID: covidwho-1054048

ABSTRACT

The COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/µl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.


Subject(s)
/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/isolation & purification , /genetics , Clinical Laboratory Techniques/methods , Diagnostic Techniques and Procedures , Diagnostic Tests, Routine , Humans , Limit of Detection , Point-of-Care Testing , RNA, Viral/genetics , Reverse Transcription , /metabolism , Sensitivity and Specificity
19.
Eur Rev Med Pharmacol Sci ; 25(1): 503-517, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1052577

ABSTRACT

OBJECTIVE: To evaluate the diagnostic accuracy of the Food and Drug Administration Emergency Use Authorization (FDA-EUA) authorized point-of-care tests (POCTs) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). MATERIALS AND METHODS: A systematic literature search was conducted using the PubMed, Embase, and Web of Science databases for articles published till August 10, 2020. We included studies providing information regarding diagnostic test accuracy of FDA-EUA POCTs for SARS-CoV-2 detection. The methodologic quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. The review protocol is registered in the International Prospective Register of Systematic Reviews (protocol number CRD42020202248). RESULTS: We included 26 studies describing a total of 3242 samples. The summary sensitivity and specificity were 0.94 [95% confidence interval (CI): 0.88-0.97] and 1.00 (95% CI: 0.99-1.00), respectively. The area under the summary receiver operating characteristic curve was 1.00 (95% CI: 0.99-1.00). A pooled analysis based on the index test revealed a summary sensitivity and specificity of Cepheid Xpert Xpress SARS-CoV-2 [0.99 (95% CI: 0.97-1.00) and 0.99 (95% CI: 0.94-1.00, respectively)] and ID NOW COVID-19 [0.78 (95% CI: 0.74-0.82) and 1.00 (95% CI: 0.98-1.00), respectively]. CONCLUSIONS: FDA-EUA POCTs, especially molecular assays, have high sensitivity, specificity, and overall diagnostic accuracy for detecting SARS-CoV-2. If approved, FDA-EUA POCTs can provide a rapid and practical way to identify infected individuals early on and help to limit the strain on the healthcare system. However, more high-quality clinical data are required to support our results.


Subject(s)
/methods , /diagnosis , Point-of-Care Testing/standards , /isolation & purification , Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , Humans , Quality Assurance, Health Care , Sensitivity and Specificity , United States , United States Food and Drug Administration
20.
Sci Rep ; 11(1): 1873, 2021 01 21.
Article in English | MEDLINE | ID: covidwho-1043028

ABSTRACT

The development of alternative isothermal amplification assays including multiple cross displacement amplification (MCDA) may address speed and portability limitations of real-time PCR (rt-PCR) methods for SARS-CoV-2 detection. We developed a novel SARS-CoV-2 MCDA assay and compared its speed and sensitivity to loop-mediated isothermal amplification (LAMP) and rt-PCR. Two MCDA assays targeting SARS-CoV-2 N gene and ORF1ab were designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and rt-PCR using DNA standards and transcribed RNA. For the N gene, MCDA was faster than LAMP and rt-PCR by 10 and 20 min, respectively with fastest time to detection at 5.2 min. rt-PCR had the highest sensitivity with the limit of detection at 10 copies/µl compared with MCDA (100 copies/µl) and LAMP (500 copies/µl). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 min, respectively. LAMP was more sensitive for ORF1ab detection with 50 copies/µl compared to MCDA (500 copies/µl). In conclusion, different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for SARS-CoV-2 while rt-PCR is the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.


Subject(s)
/diagnosis , Nucleic Acid Amplification Techniques/methods , /isolation & purification , Biological Assay/methods , /methods , Clinical Laboratory Techniques/methods , Humans , Molecular Diagnostic Techniques/methods , Phosphoproteins/genetics , Polyproteins/genetics , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Viral Proteins/genetics
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