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1.
Parasitol Res ; 121(7): 1867-1885, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-2174153

ABSTRACT

Malaria control measures have been in use for years but have not completely curbed the spread of infection. Ultimately, global elimination is the goal. A major playmaker in the various approaches to reaching the goal is the issue of proper diagnosis. Various diagnostic techniques were adopted in different regions and geographical locations over the decades, and these have invariably produced diverse outcomes. In this review, we looked at the various approaches used in malaria diagnostics with a focus on methods favorably used during pre-elimination and elimination phases as well as in endemic regions. Microscopy, rapid diagnostic testing (RDT), loop-mediated isothermal amplification (LAMP), and polymerase chain reaction (PCR) are common methods applied depending on prevailing factors, each with its strengths and limitations. As the drive toward the elimination goal intensifies, the search for ideal, simple, fast, and reliable point-of-care diagnostic tools is needed more than ever before to be used in conjunction with a functional surveillance system supported by the ideal vaccine.


Subject(s)
Malaria, Falciparum , Malaria , Diagnostic Tests, Routine/methods , Goals , Humans , Malaria/diagnosis , Malaria/prevention & control , Malaria, Falciparum/epidemiology , Microscopy/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Polymerase Chain Reaction/methods , Sensitivity and Specificity
2.
Sci Rep ; 12(1): 20282, 2022 Nov 24.
Article in English | MEDLINE | ID: covidwho-2133623

ABSTRACT

Since Coronavirus Disease-2019 (COVID-19) outbreak was reported, many commercial Nucleic Acid Amplification Tests (NAAT) have been developed all over the world, and it has been the standard method. Even though several assays were rapidly developed and applied to laboratory diagnostic testing, the performance of these assays was not evaluated in different contexts. Thus, this study aimed to assess the performance of Abbott SARS-CoV-2, Daan Gene, BGI and Sansure Biotech assays by using Composite Reference Standard (CRS). The study was conducted at the Ethiopian Public Health Institute (EPHI) from December 1 to 30/2020. Of the 164 nasopharyngeal samples were extracted by using a QIAamp RNA mini kit and Abbott DNA sample preparation system. Out of 164 samples, 59.1% were positive and 40.9% were negative by CRS. Sansure Biotech positivity was significantly low compared to CRS (p < 0.05). The overall agreement of the four assays compared to CRS was 96.3-100%. The performance of the four assays had almost comparable diagnostic performance, except for a low positive rate of Sansure Biotech assay. Hence, Sansure Biotech assay [Research Use Only (RUO)] needs further verification on its use in Ethiopia. Finally an additional study should be considered for evaluating assays with respective manufacturer claims.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Ethiopia/epidemiology , COVID-19/diagnosis , COVID-19/epidemiology , Nucleic Acid Amplification Techniques , Reference Standards
3.
Nat Commun ; 13(1): 6480, 2022 Oct 29.
Article in English | MEDLINE | ID: covidwho-2115515

ABSTRACT

Fast, inexpensive, and multiplexed detection of multiple nucleic acids is of great importance to human health, yet it still represents a significant challenge. Herein, we propose a nucleic acid testing platform, named MiCaR, which couples a microfluidic device with CRISPR-Cas12a and multiplex recombinase polymerase amplification. With only one fluorescence probe, MiCaR can simultaneously test up to 30 nucleic acid targets through microfluidic space coding. The detection limit achieves 0.26 attomole, and the multiplexed assay takes only 40 min. We demonstrate the utility of MiCaR by efficiently detecting the nine HPV subtypes targeted by the 9-valent HPV vaccine, showing a sensitivity of 97.8% and specificity of 98.1% in the testing of 100 patient samples at risk for HPV infection. Additionally, we also show the generalizability of our approach by successfully testing eight of the most clinically relevant respiratory viruses. We anticipate this effective, undecorated and versatile platform to be widely used in multiplexed nucleic acid detection.


Subject(s)
Nucleic Acids , Recombinases , Humans , CRISPR-Cas Systems/genetics , Microfluidics , Nucleic Acid Amplification Techniques , Nucleotidyltransferases
5.
Travel Med Infect Dis ; 50: 102459, 2022.
Article in English | MEDLINE | ID: covidwho-2118175

ABSTRACT

Monkeypox is an emerging zoonotic disease caused by monkeypox virus which is a DNA virus. The virus is transmitted to humans as a result of close contact with infected animals, infected humans or contaminated inanimate objects. The disease has a incubation period usually 7-14 days and it causes fever, headache, fatigue, myalgia, widespread body aches, swelling in lymph nodes and skin lesions. It may be difficult to distinguish monkeypox on the basis of clinical presentation alone, especially for cases with an atypical appearance, because of the various conditions that cause skin rashes. Testing should be offered to anyone who falls under the suspected case definition for monkeypox infection. Suitable samples are surface lesion and/or skin materials such as exudates swabs and crusts. Laboratory confirmation of specimens from suspected case is done using nucleic acid amplification testing, such as real-time or conventional polymerase chain reaction. Confirmation of MPXV infection should consider clinical and epidemiological information. Positive detection using an OPXV PCR assay followed by confirmation of MPXV via PCR and/or sequencing, or positive detection using MPXV PCR assay in suspected cases indicates confirmation of MPXV infection. Genetic sequence data (GSD) provide information on the origin and epidemic and characteristics of cases. There is a need to develop a more global and effective laboratory network for this emerging zoonosis, as well as to strengthen laboratory capacity, and international specimens referral capacities.


Subject(s)
Monkeypox virus , Monkeypox , Animals , Humans , Monkeypox virus/genetics , Monkeypox/diagnosis , Monkeypox/epidemiology , Monkeypox/pathology , Polymerase Chain Reaction , Nucleic Acid Amplification Techniques
6.
Biosensors (Basel) ; 12(11)2022 Nov 08.
Article in English | MEDLINE | ID: covidwho-2109935

ABSTRACT

Worldwide infection due to SARS-CoV-2 revealed that short-time and extremely high-sensitivity detection of nucleic acids is a crucial technique for human beings. Polymerase chain reactions have been mainly used for the SARS-CoV-2 detection over the years. However, an advancement in quantification of the detection and shortening runtime is important for present and future use. Here, we report a rapid detection scheme that is a combination of nucleic acid amplification and a highly efficient fluorescence biosensor, that is, a metasurface biosensor composed of a pair of an all-dielectric metasurface and a microfluidic transparent chip. In the present scheme, we show a series of proof-of-concept experimental results that the metasurface biosensors detected amplicons originating from attomolar SARS-CoV-2 nucleic acids and that the amplification was implemented within 1 h. Furthermore, this detection capability substantially satisfies an official requirement of 100 RNA copies/140 µL, which is a criterion for the reliable infection tests.


Subject(s)
Biosensing Techniques , COVID-19 , Nucleic Acids , Humans , SARS-CoV-2 , COVID-19/diagnosis , Sensitivity and Specificity , Nucleic Acid Amplification Techniques/methods , Molecular Diagnostic Techniques/methods
7.
Nature ; 611(7936): 570-577, 2022 11.
Article in English | MEDLINE | ID: covidwho-2106425

ABSTRACT

Expanding our global testing capacity is critical to preventing and containing pandemics1-9. Accordingly, accessible and adaptable automated platforms that in decentralized settings perform nucleic acid amplification tests resource-efficiently are required10-14. Pooled testing can be extremely efficient if the pooling strategy is based on local viral prevalence15-20; however, it requires automation, small sample volume handling and feedback not available in current bulky, capital-intensive liquid handling technologies21-29. Here we use a swarm of millimetre-sized magnets as mobile robotic agents ('ferrobots') for precise and robust handling of magnetized sample droplets and high-fidelity delivery of flexible workflows based on nucleic acid amplification tests to overcome these limitations. Within a palm-sized printed circuit board-based programmable platform, we demonstrated the myriad of laboratory-equivalent operations involved in pooled testing. These operations were guided by an introduced square matrix pooled testing algorithm to identify the samples from infected patients, while maximizing the testing efficiency. We applied this automated technology for the loop-mediated isothermal amplification and detection of the SARS-CoV-2 virus in clinical samples, in which the test results completely matched those obtained off-chip. This technology is easily manufacturable and distributable, and its adoption for viral testing could lead to a 10-300-fold reduction in reagent costs (depending on the viral prevalence) and three orders of magnitude reduction in instrumentation cost. Therefore, it is a promising solution to expand our testing capacity for pandemic preparedness and to reimagine the automated clinical laboratory of the future.


Subject(s)
Automation , COVID-19 Testing , Magnets , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Robotics , SARS-CoV-2 , Humans , COVID-19/diagnosis , COVID-19/virology , COVID-19 Testing/methods , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/methods , Pandemics/prevention & control , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Algorithms , Automation/economics , Automation/methods , Robotics/methods , Indicators and Reagents/economics
8.
Biosensors (Basel) ; 12(11)2022 Nov 07.
Article in English | MEDLINE | ID: covidwho-2099353

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 has drawn attention to the need for fast and accurate diagnostic testing. Concerns from emerging SARS-CoV-2 variants and other circulating respiratory viral pathogens further underscore the importance of expanding diagnostic testing to multiplex detection, as single-plex diagnostic testing may fail to detect emerging variants and other viruses, while sequencing can be too slow and too expensive as a diagnostic tool. As a result, there have been significant advances in multiplex nucleic-acid-based virus diagnostic testing, creating a need for a timely review. This review first introduces frequent nucleic acid targets for multiplex virus diagnostic tests, then proceeds to a comprehensive and up-to-date overview of multiplex assays that incorporate various detection reactions and readout modalities. The performances, advantages, and disadvantages of these assays are discussed, followed by highlights of platforms that are amenable for point-of-care use. Finally, this review points out the remaining technical challenges and shares perspectives on future research and development. By examining the state of the art and synthesizing existing development in multiplex nucleic acid diagnostic tests, this review can provide a useful resource for facilitating future research and ultimately combating COVID-19.


Subject(s)
COVID-19 , Nucleic Acids , Humans , SARS-CoV-2 , COVID-19/diagnosis , Pandemics , Diagnostic Tests, Routine , Nucleic Acid Amplification Techniques , Sensitivity and Specificity
9.
Biotechniques ; 73(5): 247-255, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2098927

ABSTRACT

Loop-mediated isothermal amplification (LAMP) has proven a robust and reliable nucleic acid amplification method that is well suited for simplified and rapid molecular diagnostics. Various approaches have emerged for sequence-specific detection of LAMP products, but with limitations to their widespread utility or applicability for single-nucleotide polymorphism detection and multiplexing. Here we demonstrate the use of simple hybridization probes (as used for qPCR) that enable simple multiplexing and SARS-CoV-2 variant typing in reverse-transcription LAMP. This approach requires no modification to the LAMP primers and is amenable to the detection of single-nucleotide polymorphisms and small sequence changes, which is usually difficult in LAMP. By extending LAMP's ability to be utilized for multitarget and single-base change detection, we hope to increase its potential to enable more and better molecular diagnostic testing.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Sensitivity and Specificity , Nucleic Acid Amplification Techniques/methods , Molecular Diagnostic Techniques/methods , RNA, Viral
10.
Methods ; 203: 431-446, 2022 07.
Article in English | MEDLINE | ID: covidwho-2096167

ABSTRACT

Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas3, Cas9, Cas12, Cas13, Cas14 etc.) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, lateral flow assay detection and other. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, CARMEN or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing COVID-19) and outbreaks of zoonotic viruses (African Swine Fever Virus etc.) urgently need the developing and distribution of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.


Subject(s)
African Swine Fever Virus , COVID-19 , Communicable Diseases , Animals , COVID-19/diagnosis , COVID-19/epidemiology , CRISPR-Cas Systems/genetics , Communicable Diseases/diagnosis , Communicable Diseases/genetics , Humans , Nucleic Acid Amplification Techniques/methods , Point-of-Care Systems , Swine
12.
Int J Mol Sci ; 23(21)2022 Oct 28.
Article in English | MEDLINE | ID: covidwho-2090209

ABSTRACT

Consistently emerging variants and the life-threatening consequences of SARS-CoV-2 have prompted worldwide concern about human health, necessitating rapid and accurate point-of-care diagnostics to limit the spread of COVID-19. Still, However, the availability of such diagnostics for COVID-19 remains a major rate-limiting factor in containing the outbreaks. Apart from the conventional reverse transcription polymerase chain reaction, loop-mediated isothermal amplification-based (LAMP) assays have emerged as rapid and efficient systems to detect COVID-19. The present study aims to develop RT-LAMP-based assay system for detecting multiple targets in N, ORF1ab, E, and S genes of the SARS-CoV-2 genome, where the end-products were quantified using spectrophotometry, paper-based lateral-flow devices, and electrochemical sensors. The spectrophotometric method shows a LOD of 10 agµL-1 for N, ORF1ab, E genes and 100 agµL-1 for S gene in SARS-CoV-2. The developed lateral-flow devices showed an LOD of 10 agµL-1 for all four gene targets in SARS-CoV-2. An electrochemical sensor developed for N-gene showed an LOD and E-strip sensitivity of log 1.79 ± 0.427 pgµL-1 and log 0.067 µA/pg µL-1/mm2, respectively. The developed assay systems were validated with the clinical samples from COVID-19 outbreaks in 2020 and 2021. This multigene target approach can effectively detect emerging COVID-19 variants using combination of various analytical techniques at testing facilities and in point-of-care settings.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , Sensitivity and Specificity , Nucleic Acid Amplification Techniques/methods , Molecular Diagnostic Techniques/methods , RNA, Viral/genetics
13.
Mol Med ; 28(1): 40, 2022 04 09.
Article in English | MEDLINE | ID: covidwho-2089157

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already caused 6 million deaths worldwide. While asymptomatic individuals are responsible of many potential transmissions, the difficulty to identify and isolate them at the high peak of infection constitutes still a real challenge. Moreover, SARS-CoV-2 provokes severe vascular damage and thromboembolic events in critical COVID-19 patients, deriving in many related deaths and long-hauler symptoms. Understanding how these processes are triggered as well as the potential long-term sequelae, even in asymptomatic individuals, becomes essential. METHODS: We have evaluated, by application of a proteomics-based quantitative approach, the effect of serum from COVID-19 asymptomatic individuals over circulating angiogenic cells (CACs). Healthy CACs were incubated ex-vivo with the serum of either COVID-19 negative (PCR -/IgG -, n:8) or COVID-19 positive asymptomatic donors, at different infective stages: PCR +/IgG - (n:8) and PCR -/IgG + (n:8). Also, a label free quantitative approach was applied to identify and quantify protein differences between these serums. Finally, machine learning algorithms were applied to validate the differential protein patterns in CACs. RESULTS: Our results confirmed that SARS-CoV-2 promotes changes at the protein level in the serum of infected asymptomatic individuals, mainly correlated with altered coagulation and inflammatory processes (Fibrinogen, Von Willebrand Factor, Thrombospondin-1). At the cellular level, proteins like ICAM-1, TLR2 or Ezrin/Radixin were only up-regulated in CACs treated with the serum of asymptomatic patients at the highest peak of infection (PCR + /IgG -), but not with the serum of PCR -/IgG + individuals. Several proteins stood out as significantly discriminating markers in CACs in response to PCR or IgG + serums. Many of these proteins particiArticle title: Kindly check and confirm the edit made in the article title.pate in the initial endothelial response against the virus. CONCLUSIONS: The ex vivo incubation of CACs with the serum of asymptomatic COVID-19 donors at different stages of infection promoted protein changes representative of the endothelial dysfunction and inflammatory response after viral infection, together with activation of the coagulation process. The current approach constitutes an optimal model to study the response of vascular cells to SARS-CoV-2 infection, and an alternative platform to test potential inhibitors targeting either the virus entry pathway or the immune responses following SARS-CoV-2 infection.


Subject(s)
COVID-19 , Humans , Immunoglobulin G , Nucleic Acid Amplification Techniques , SARS-CoV-2
14.
ACS Appl Mater Interfaces ; 14(45): 50534-50542, 2022 Nov 16.
Article in English | MEDLINE | ID: covidwho-2087120

ABSTRACT

The CRISPR-Cas system was developed into a molecular diagnostic tool with high sensitivity, low cost, and high specificity in recent years. Colorimetric assays based on nanozymes offer an attractive point-of-care testing method for their low cost of use and user-friendly operation. Here, a MnO2 nanozyme-mediated CRISPR-Cas12a system was instituted to detect SARS-CoV-2. MnO2 nanorods linked to magnetic beads via a single-stranded DNA (ssDNA) linker used as an oxidase-like nanozyme inducing the color change of 3,3',5,5'-tetramethylbenzidine, which can be distinguished by the naked eye. The detection buffer color will change when the Cas12a is activated by SARS-CoV-2 and indiscriminately cleave the linker ssDNA. The detection limit was 10 copies per microliter and showed no cross-reaction with other coronaviruses. The nanozyme-mediated CRISPR-Cas12a system shows high selectivity and facile operation, with great potential for molecular diagnosis in point-of-care testing applications.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , CRISPR-Cas Systems/genetics , Manganese Compounds , COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , Oxides , DNA, Single-Stranded
15.
Anal Chem ; 94(44): 15472-15480, 2022 Nov 08.
Article in English | MEDLINE | ID: covidwho-2087112

ABSTRACT

Integrated clustered regularly interspaced short palindromic repeat (CRISPR)-loop-mediated amplification (LAMP) technology is of great importance in CRISPR-based diagnostic systems, which urgently needs to be developed to improve diagnostic accuracy. A labor-free, contamination-free, and fully automated droplet manipulation platform for the CRISPR-LAMP technology has not been developed before. Herein, we propose a fully automated CRISPR-LAMP platform, which can precisely manipulate the CRISPR-LAMP droplet and perform combined reactions with high sensitivity and specificity. SARS-CoV-2 Spike T478K, D614G, P681R, and P681H mutations, typical point mutations of B.1.617.2 (Delta) and Omicron variants, are monitored with this platform with a detection limit of 102 copies/µL. Allele discrimination between the mutants and wild type is significant with the designed one/two-mismatch CRISPR RNA (crRNA) at a limit of 102 copies/µL. Chemically synthesized and modified crRNAs greatly increase the CRISPR-LAMP signal, which advance the wide application. Combined with the previously developed RdRp CRISPR-LAMP assay, clinical results showed that Spike T478K and P681H can discriminate the mutant type form the wild type with 70% (49.66-85.50%, 95% confidence interval) and 78% (57.27-90.62%, 95% confidence interval) sensitivity, respectively, and 100% specificity (51.68-100%, 95% confidence interval), and the RdRp target can detect SARS-CoV-2 strains with 85% sensitivity (65.39-95.14%, 95% confidence interval) and 100% specificity (51.68-100%, 95% confidence interval). We believe that this automatic digital microfluid (DMF) system can advance the integrated CRISPR-LAMP technology with higher stability, sensitivity, and practicability, also for other CRISPR-associated diagnostic platforms.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , RNA-Dependent RNA Polymerase , Sensitivity and Specificity
16.
Anal Biochem ; 659: 114960, 2022 Dec 15.
Article in English | MEDLINE | ID: covidwho-2085839

ABSTRACT

COVID-19 pandemic highlighted the demand for the fast and reliable detection of viral RNA. Although various methods for RNA amplification and detection have been proposed, some limitations, including those caused by reverse transcription (RT), need to be overcome. Here, we report on the direct detection of specific RNA by conventional polymerase chain reaction (PCR) requiring no prior RT step. It was found that Hemo KlenTaq (HKTaq), which is posed as DNA-dependent DNA polymerase, possesses reverse transcriptase activity and provides reproducible amplification of RNA targets with an efficiency comparable to common RT-PCR. Using nasopharyngeal swab extracts from COVID-19-positive patients, the high reliability of SARS-CoV-2 detection based on HKTaq was demonstrated. The most accurate detection of specific targets are provided by nearby primers, which allow to determine RNA in solutions affected to multiple freeze-thaw cycles. HKTaq can be used for elaboration of simplified amplification techniques intended for the analysis of any specific RNA and requiring only one DNA polymerase.


Subject(s)
COVID-19 , RNA, Viral , Humans , Clinical Laboratory Techniques/methods , COVID-19 Testing , Nucleic Acid Amplification Techniques/methods , Pandemics , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , RNA, Viral/genetics , RNA, Viral/analysis , RNA-Directed DNA Polymerase/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Taq Polymerase/metabolism
17.
JAMA Netw Open ; 5(10): e2236288, 2022 10 03.
Article in English | MEDLINE | ID: covidwho-2059208

ABSTRACT

Importance: Early and accurate diagnostic testing for SARS-CoV-2 is essential to initiate appropriate treatment and infection control and prevention measures among patients presenting to the hospital. Objective: To evaluate the diagnostic sensitivity of the SARS-CoV-2 nucleic acid amplification test (NAAT) performed within 24 hours of arrival to the emergency department among a nationally representative sample of patients. Design, Setting, and Participants: This diagnostic study was conducted at 47 hospitals across 7 provinces in Canada participating in the Canadian COVID-19 Rapid Response Emergency Department Network among consecutive eligible patients presenting to a participating emergency department who were tested for SARS-CoV-2 from March 1, 2020, to December 31, 2021. Patients not tested within 24 hours of arrival and those presenting with a positive result from a test performed in the community were excluded. Main Outcomes and Measures: The primary outcome was a positive result from the SARS-CoV-2 NAAT. Outcome measures were the diagnostic sensitivity and yield of the SARS-CoV-2 NAAT. Results: Of 132 760 eligible patients (66 433 women [50.0%]; median age, 57 years [IQR, 37-74 years]), 17 174 (12.9%) tested positive for SARS-CoV-2 within 14 days of their first NAAT. The diagnostic sensitivity of the SARS-CoV-2 NAAT was 96.2% (17 070 of 17 740 [95% CI, 95.9%-96.4%]) among all of the tests performed. Estimates ranged from a high of 97.7% (1710 of 1751 [95% CI, 96.8%-98.3%]) on day 2 of symptoms to a low of 90.4% (170 of 188 [95% CI, 85.3%-94.2%]) on day 11 of symptoms among patients presenting with COVID-19 symptoms. Among patients reporting COVID-19 symptoms, the sensitivity of the SARS-CoV-2 NAAT was 97.1% (11 870 of 12 225 [95% CI, 96.7%-97.3%]) compared with 87.6% (812 of 927 [95% CI, 85.2%-89.6%]) among patients without COVID-19 symptoms. The diagnostic yield of the SARS-CoV-2 NAAT was 12.0% (18 985 of 158 004 [95% CI, 11.8%-12.2%]) and varied from a high of 20.0% (445 of 2229 [95% CI, 18.3%-21.6%]) among patients tested on day 10 after symptom onset to a low of 8.1% (1686 of 20 719 [95% CI, 7.7%-8.5%]) among patients presenting within the first 24 hours of symptom onset. Conclusions and Relevance: This study suggests that the diagnostic sensitivity was high for the first SARS-CoV-2 NAAT performed in the hospital and did not vary significantly by symptom duration. Repeated testing of patients with negative test results should be avoided unless their pretest probability of disease is high.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Canada , Female , Hospitals , Humans , Middle Aged , Nucleic Acid Amplification Techniques
18.
Se Pu ; 40(9): 773-781, 2022 Sep.
Article in Chinese | MEDLINE | ID: covidwho-2055456

ABSTRACT

The rapid global spread of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has introduced various challenges in global public health systems. The poor applicability and sensitivity of the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and antigen-based tests, as well as the persistent emergence of SARS-CoV-2 variants with different mutations hinder satisfactory epidemic prevention and control. Therefore, there is an urgent need for diagnostic technologies capable of distinguishing SARS-CoV-2 variants with high sensitivity and low (or no) equipment dependence. Diagnosis based on clustered regularly interspaced short palindromic repeats (CRISPR) has low equipment requirements and is programmable, sensitive, and easy to use. Various nucleic acid detection tools with great clinical potential have been developed for the diagnosis of infectious diseases. Therefore, this review focuses on the reported state-of-the-art CRISPR diagnostic technologies developed for the detection and differentiation of SARS-CoV-2 variants, summarizes their characteristics and provides an outlook for their development.


Subject(s)
COVID-19 , Nucleic Acids , COVID-19/diagnosis , CRISPR-Cas Systems , Humans , Nucleic Acid Amplification Techniques , SARS-CoV-2/genetics
19.
PLoS One ; 17(9): e0256789, 2022.
Article in English | MEDLINE | ID: covidwho-2054260

ABSTRACT

The COVID-19 pandemic has highlighted the need for broader access to molecular diagnostics. Colorimetric isothermal nucleic acid amplification assays enable simplified instrumentation over more conventional PCR diagnostic assays and, as such, represent a promising approach for addressing this need. In particular, colorimetric LAMP (loop-mediated isothermal amplification) has received a great deal of interest recently. However, there do not currently exist robust instruments for performing these kinds of assays in high throughput with real-time readout of amplification signals. To address this need, we developed LARI, the LAMP Assay Reader Instrument. We have deployed over 50 LARIs for routine use in R&D and production environments, with over 12,000 assays run to date. In this paper, we present the design and construction of LARI along with thermal, optical, and assay performance characteristics. LARI can be produced for under $1500 and has broad applications in R&D, point-of-care diagnostics, and global health.


Subject(s)
COVID-19 , Nucleic Acids , COVID-19/diagnosis , Colorimetry , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Pandemics , Sensitivity and Specificity
20.
Viruses ; 14(9)2022 09 17.
Article in English | MEDLINE | ID: covidwho-2043977

ABSTRACT

Rapid and accurate diagnosis of SARS-CoV-2 infection is essential for the management of the COVID-19 outbreak. RT-LAMP LoopDeetect COVID-19 (LoopDeescience, France) is a rapid molecular diagnostic tool which operates with the LoopDeelab (LoopDeescience, France) device. RAPID COVID is a prospective double-blind research protocol which was conducted to evaluate the concordance between Loopdeetect COVID-19 and RT-PCR Allplex 2019 n-Cov (Seegene, Korea). Between 11 May 2020 and 14 June 2021, a total of 1122 nasopharyngeal swab specimens were collected, of which 741 were finally analysed. There were 32 "positive" and "indeterminate" RT-PCR results. The intrinsic performances of Loopdeetect COVID-19 are equivalent to other commercial RT-LAMP PCR COVID-19 kits, with a sensitivity and specificity of 69.23% [CI 95%: 48.21-85.67] and 100% [CI 95%: 99.58-100.00], respectively. To the best of our knowledge, LoopDeelab is the only LAMP PCR diagnostic device allowing such a fast and reliable analysis with low-cost equipment; this makes it a new and innovative technology, designed for field use. This device being portable, the development of other detection kits will be useful for the management of epidemics with a high attack rate and would facilitate the rapid application of health measures.


Subject(s)
COVID-19 , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19 Testing , Humans , Nucleic Acid Amplification Techniques/methods , Pandemics , Prospective Studies , SARS-CoV-2/genetics , Sensitivity and Specificity
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