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1.
PLoS One ; 17(1): e0261853, 2022.
Article in English | MEDLINE | ID: covidwho-1622346

ABSTRACT

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription/genetics , SARS-CoV-2/genetics , COVID-19/virology , Feasibility Studies , Humans , Nasopharynx/virology , Pandemics/prevention & control , Sensitivity and Specificity , Serologic Tests/methods , Specimen Handling/methods
2.
Biosens Bioelectron ; 200: 113925, 2022 Mar 15.
Article in English | MEDLINE | ID: covidwho-1588208

ABSTRACT

The current COVID-19 pandemic caused by SARS-CoV-2 is raging, seriously threatening people's lives. The establishment of rapid and accurate pathogen detection technology is not only critical in this epidemic, but also a reminder that we must always be prepared for possible future outbreaks. Therefore, we developed a Palm Germ-Radar (PaGeR) device for rapid and simple detection of COVID-19 from extracted patient sample RNA by RT-LAMP. The whole procedure of rapid COVID-19 detection is based on 4 simple steps: inactivation, extraction, amplification, and detection. SARS-CoV-2 down to 1 copy/µL could be detected selectively with naked-eye. Three detection methods (colorimetric, fluorometric and lateral dipstick readout) could be performed in PaGeR instrument. By employing the PaGeR, we successfully detected SARS-CoV-2 in clinical RNA samples isolated from swab specimens. The results showed that 15 out of 17 COVID-19 patients were diagnosed as positive while all 55 normal samples were diagnosed as negative. Therefore, the developed PaGeR instrument can realize the detection of COVID-19 with easily visualized results, providing a promising instrument for rapid detection in the community as well as at home.


Subject(s)
Biosensing Techniques , COVID-19 , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Pandemics , RNA, Viral/genetics , Radar , Reverse Transcription , SARS-CoV-2 , Sensitivity and Specificity
3.
Mol Biol (Mosk) ; 55(6): 944-955, 2021.
Article in Russian | MEDLINE | ID: covidwho-1539064

ABSTRACT

A prototype of a system for the detection of infectious human pneumonia pathogens based on multiplex solid-phase reverse transcription PCR (RT-PCR) was developed. Primers were designed to identify the DNA of six bacterial pneumonia pathogen strains, and the RNA of two viral pathogens of pneumonia: influenza A and SARS-CoV-2. The signal accumulation of elongated immobilized primers occurs due to the incorporation of fluorescently labeled nucleotides in the chain. The signal is detected after all the components of the mixture are removed, which significantly reduces the background signal and increases the sensitivity of the analysis. The use of a specialized detector makes it possible to read the signals of elongated primers directly through the transparent cover film of the reaction chamber. This solution is designed to prevent cross-contamination and is suitable for simultaneous testing of a large number of test samples. The proposed platform is able to detect the presence of several pathogens of pneumonia in a sample and has an open architecture that allows expansion of the range of pathogenic bacteria and viruses that can be detected.


Subject(s)
COVID-19 , Reverse Transcription , Humans , Multiplex Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Sensitivity and Specificity
4.
BMC Infect Dis ; 21(1): 1162, 2021 Nov 17.
Article in English | MEDLINE | ID: covidwho-1523285

ABSTRACT

BACKGROUND: Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step. METHODS: In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets. RESULTS: Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9-96.1%) and 67.4% sensitive (95% CI: 51.5-80.9%) for E gene and RdRp gene, respectively. CONCLUSION: Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Reverse Transcription , Sensitivity and Specificity
5.
J Clin Microbiol ; 59(12): e0144621, 2021 11 18.
Article in English | MEDLINE | ID: covidwho-1522905

ABSTRACT

To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike, and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log10 genome equivalents (GE)/ml for the three initial targets (∼1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle (CT) values of <30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 CT values of ≥30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in the receptor binding domain, and it can be quickly modified to detect new mutations that emerge.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Mutation , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcription
6.
Clin Lab ; 67(11)2021 Nov 01.
Article in English | MEDLINE | ID: covidwho-1513105

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that brings a significant public health challenge. A rapid and simple method is necessary for testing suspected samples and screening the population. METHODS: To better monitor sample effectiveness, this study described a method to detect nucleocapsid protein gene (N gene) of SARS-CoV-2 and human ACTB gene employing real-time duplex reverse transcription multienzyme isothermal rapid amplification (RT-MIRA) assays. RESULTS: The established real-time duplex RT-MIRA assays showed that no cross-reactions were observed to other pathogens and the detection limit was 100 copies/reaction. Using simulated clinical samples to test established assays further and the amplification process took no more than 20 minutes at 42°C. CONCLUSIONS: RT-MIRA assays are faster and easier than reverse transcription real-time polymerase chain reaction (RT-PCR). It is expected to be further optimized and evaluated in the detection of SARS-CoV-2 confirmed cases.


Subject(s)
COVID-19 , Reverse Transcription , Humans , Nucleic Acid Amplification Techniques , SARS-CoV-2 , Sensitivity and Specificity
7.
Front Cell Infect Microbiol ; 11: 678703, 2021.
Article in English | MEDLINE | ID: covidwho-1505766

ABSTRACT

In December 2019, a severe acute respiratory syndrome caused by SARS-CoV-2 spread rapidly worldwide. Portable nucleic acid tests of SARS-CoV-2 are critically important for diagnostics. In this study, we used an isothermal amplification method-Multienzyme Isothermal Rapid Amplification (MIRA)-for rapid detection of SARS-CoV-2. We designed the primers and probes in ORF1ab and N gene of SARS-CoV-2. The amplicons could be monitored by lateral flow dipsticks (LFDs). The reaction temperature, time, concentrations of primers and probes, and working volume were optimized. Four commercial swab collection buffers were used to test the amplification efficacy of our assay without RNA extraction. Our assay was able to amplify duplex targets of SARS-CoV-2 in one single reaction using one-step RT-MIRA. The assay worked well in a low volume of 10 µl at 38°C for 20 min. Using three collection buffers without guanidinium, our assay was able to amplify efficaciously without RNA extraction. The 95% limit of detection (LoD) of the RT-MIRA assay was 49.5 (95% CI, 46.8-52.7) copies/ml for ORF1ab gene and 48.8 (95% CI, 46.5-52.6) copies/ml for N gene. There is no cross-reaction with other human respiratory pathogens, such as SARS-CoV, MERS-CoV, influenza A virus, influenza B virus, human adenovirus, respiratory syncytial virus, human parainfluenza virus, and coronavirus 229E in our assay. The precision evaluation revealed that the C50-20% to C50+20% range bounds the C5-C95 interval. This assay also showed high anti-interference ability. The extraction-free RT-MIRA and qPCR detection results of 243 nucleic acid specimens from suspected patients or national references showed a 100.0% (95% confidence interval, 94.2%-100.0%) positive predictive value and a 100.0% (95% confidence interval, 92.7%-100.0%) negative predictive value. Compared with qPCR, the kappa value of the two assays was 1.00 (P < 0.0001). In conclusion, we provide a portable and visualized method for detection of SARS-CoV-2 without RNA extraction, allowing its application in SARS-CoV-2 on-site detection.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Nucleic Acid Amplification Techniques , Point-of-Care Testing , RNA, Viral/genetics , Reverse Transcription
8.
Clin Infect Dis ; 73(9): e3102-e3105, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1500983

ABSTRACT

This study analyzed the cerebrospinal fluid features of 31 coronavirus disease 2019 (COVID-19) patients with neurological complications. We observed neither severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the cerebrospinal fluid, nor intrathecal immunoglobulin G (IgG) synthesis but did observe signs of blood-brain barrier disruption. These results might serve as a basis for a better understanding of SARS-CoV-2 related neuropathogenesis.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Immunoglobulin G , Reverse Transcriptase Polymerase Chain Reaction , Reverse Transcription
9.
Sci Rep ; 11(1): 21385, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1493218

ABSTRACT

Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen's kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar's test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/epidemiology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , Genome, Viral , Humans , Infection Control/methods , Limit of Detection , Mass Screening/methods , RNA, Viral/genetics , RNA, Viral/isolation & purification , RNA-Directed DNA Polymerase/genetics , Reproducibility of Results , Reverse Transcription/genetics , Sensitivity and Specificity
10.
J Clin Microbiol ; 59(7): e0037421, 2021 06 18.
Article in English | MEDLINE | ID: covidwho-1486479

ABSTRACT

We evaluated the quantitative DiaSorin Liaison severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test in symptomatic and asymptomatic individuals consulting their general practitioners (GPs) during a period of stable intense virus circulation (213/100,000 habitants per day). Leftover reverse transcription-PCR (RT-PCR) positive (n = 204) and negative (n = 210) nasopharyngeal samples were randomly selected among fresh routine samples collected from patients consulting their GPs. Samples were tested on Liaison XL according to the manufacturer's instructions. Equivocal results were considered negative. The overall sensitivity and specificity of the Liaison antigen test compared to RT-PCR were 65.7% (95% confidence interval [CI], 58.9% to 71.9%) and 100% (CI, 97.8% to 100%). Sensitivity in samples with viral loads of ≥105, ≥104, and ≥103 copies/ml were 100% (CI, 96.3% to 100.0%), 96.5% (CI, 91.8% to 98.7%), and 87.4% (CI, 81.3% to 91.5%), respectively. All samples with ≤103 copies/ml were antigen negative. The ratio of antigen concentration to viral load in samples with ≥103 copies/ml was comparable in symptomatic and asymptomatic individuals (P = 0.58). The proportion of RT-PCR-positive participants with a high viral load (≥105 copies/ml) was not significantly higher in symptomatic than in asymptomatic participants (63.9% [CI, 54.9% to 72.0%] versus 51.9% [CI, 41.1% to 62.6%]; P = 0.11), but the proportion of participants with a low viral load (<103 copies/ml) was significantly higher in asymptomatic than in symptomatic RT-PCR-positive participants (35.4% [CI, 25.8% to 46.4%] versus 14.3% [CI, 9.0% to 21.8%]; P < 0.01). Sensitivity and specificity in samples with a viral load of ≥104 copies/ml were 96.5% and 100%. The correlation of antigen concentration with viral load was comparable in symptomatic and asymptomatic individuals.


Subject(s)
COVID-19 , Humans , Outpatients , Real-Time Polymerase Chain Reaction , Reverse Transcription , SARS-CoV-2 , Sensitivity and Specificity , Viral Load
11.
Clin Infect Dis ; 73(6): e1348-e1355, 2021 09 15.
Article in English | MEDLINE | ID: covidwho-1479943

ABSTRACT

BACKGROUND: Real-time reverse transcription polymerase chain reaction (rRT-PCR) and antigen tests are important diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sensitivity of antigen tests has been shown to be lower than that of rRT-PCR; however, data to evaluate epidemiologic characteristics that affect test performance are limited. METHODS: Paired mid-turbinate nasal swabs were collected from university students and staff and tested for SARS-CoV-2 using both Quidel Sofia SARS Antigen Fluorescent Immunoassay (FIA) and rRT-PCR assay. Specimens positive by either rRT-PCR or antigen FIA were placed in viral culture and tested for subgenomic RNA (sgRNA). Logistic regression models were used to evaluate characteristics associated with antigen results, rRT-PCR cycle threshold (Ct) values, sgRNA, and viral culture. RESULTS: Antigen FIA sensitivity was 78.9% and 43.8% among symptomatic and asymptomatic participants, respectively. Among rRT-PCR positive participants, negative antigen results were more likely among asymptomatic participants (odds ratio [OR] 4.6, 95% confidence interval [CI]: 1.3-15.4) and less likely among participants reporting nasal congestion (OR 0.1, 95% CI: .03-.8). rRT-PCR-positive specimens with higher Ct values (OR 0.5, 95% CI: .4-.8) were less likely, and specimens positive for sgRNA (OR 10.2, 95% CI: 1.6-65.0) more likely, to yield positive virus isolation. Antigen testing was >90% positive in specimens with Ct values < 29. Positive predictive value of antigen test for positive viral culture (57.7%) was similar to that of rRT-PCR (59.3%). CONCLUSIONS: SARS-CoV-2 antigen test advantages include low cost, wide availability and rapid turnaround time, making them important screening tests. The performance of antigen tests may vary with patient characteristics, so performance characteristics should be accounted for when designing testing strategies and interpreting results.


Subject(s)
COVID-19 , SARS-CoV-2 , Antigens, Viral , Humans , RNA , Reverse Transcriptase Polymerase Chain Reaction , Reverse Transcription , Sensitivity and Specificity , Universities
14.
J Infect Chemother ; 28(1): 120-123, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1433521

ABSTRACT

INTRODUCTION: The pandemic of a novel coronavirus disease 2019 (COVID-19) caused by a severe acute respiratory coronavirus 2 (SARS-CoV-2) infection has been problematic worldwide. A new SARS-CoV-2 diagnostic test (SmartAmp) was licensed in Japan in July 2021. This method, which enables us to diagnose COVID-19 as well as a gene mutation on the virus, is promising to reduce medical costs and staff labor. PATIENTS AND METHODS: To analyze the diagnostic accuracy of the SmartAmp assay for diagnosing COVID-19, we performed this retrospective study at our institute during April and May 2021. We compared the results of the SmartAmp assay and real-time reverse transcription-polymerase chain reaction (rRT-PCR) using a saliva sample from individuals suspected as having COVID-19. RESULTS: Out of 70 samples tested, the SmartAmp assay had 50 (71%) positive and 20 (29%) negative results. Using rRT-PCR as a reference, the diagnostic accuracy displayed a sensitivity of 84%, a specificity of 95%, a positive predictive value of 97.7%, and a negative predictive value of 70.4%. On the other hand, false-negative cases were found in 7 (10%), and there was no significant difference of Ct-value between true positive and false negative cases (Mean Ct-value 25.2 vs. 27.5 cycles, p = 0.226 by Mann-Whitney U test). CONCLUSION: The SmartAmp assay is a valuable method to diagnose COVID-19 rapidly. However, the negative predictive value is not high enough to diagnose the disease, so that negative results should be considered for rRT-PCR testing if patients are suspected of having COVID-19.


Subject(s)
COVID-19 , Saliva , Humans , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction , Reverse Transcription , SARS-CoV-2 , Sensitivity and Specificity
15.
Anal Chem ; 93(37): 12808-12816, 2021 09 21.
Article in English | MEDLINE | ID: covidwho-1402013

ABSTRACT

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.


Subject(s)
COVID-19 , Reverse Transcription , COVID-19 Testing , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Recombinases/genetics , SARS-CoV-2 , Sensitivity and Specificity , Technology
16.
Viruses ; 13(2)2021 02 10.
Article in English | MEDLINE | ID: covidwho-1395005

ABSTRACT

Since the discovery of HIV-1, the viral capsid has been recognized to have an important role as a structural protein that holds the viral genome, together with viral proteins essential for viral life cycle, such as the reverse transcriptase (RT) and the integrase (IN). The reverse transcription process takes place between the cytoplasm and the nucleus of the host cell, thus the Reverse Transcription Complexes (RTCs)/Pre-integration Complexes (PICs) are hosted in intact or partial cores. Early biochemical assays failed to identify the viral CA associated to the RTC/PIC, possibly due to the stringent detergent conditions used to fractionate the cells or to isolate the viral complexes. More recently, it has been observed that some host partners of capsid, such as Nup153 and CPSF6, can only bind multimeric CA proteins organized in hexamers. Those host factors are mainly located in the nuclear compartment, suggesting the entrance of the viral CA as multimeric structure inside the nucleus. Recent data show CA complexes within the nucleus having a different morphology from the cytoplasmic ones, clearly highlighting the remodeling of the viral cores during nuclear translocation. Thus, the multimeric CA complexes lead the viral genome into the host nuclear compartment, piloting the intranuclear journey of HIV-1 in order to successfully replicate. The aim of this review is to discuss and analyze the main discoveries to date that uncover the viral capsid as a key player in the reverse transcription and PIC maturation until the viral DNA integration into the host genome.


Subject(s)
Capsid/metabolism , Cell Nucleus/virology , HIV-1/physiology , Active Transport, Cell Nucleus , Capsid/chemistry , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Cell Nucleus/metabolism , HIV-1/chemistry , HIV-1/metabolism , Models, Biological , Nuclear Pore Complex Proteins/metabolism , Reverse Transcription , Virus Integration , Virus Replication
18.
Jpn J Infect Dis ; 74(4): 325-332, 2021 Jul 21.
Article in English | MEDLINE | ID: covidwho-1380108

ABSTRACT

Studies describing reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay-based infection control strategies (LAMP-based ICSs) for coronavirus disease 2019 (COVID-19) are limited. We reviewed the medical records of cases in which RT-LAMP was performed. Standard ICSs and LAMP-based ICSs were implemented during the study period. The strategies were intended to impose longer periods of infection control precautions (ICPs) for specific patients, such as those with a history of exposure to COVID-19 patients and/or bilateral ground glass opacities (bGGO) on chest computed tomography (CT). Of 212 patients, which included 13 confirmed COVID-19 patients in the diagnostic cohort, exposure to COVID-19 patients (P <0.0001) and chest CT bGGO (P = 0.0022) were identified as significant predictors of COVID-19. In the 173 hospitalized patients in which the results of the first RT-LAMP were negative, the duration of ICPs was significantly longer in patients with exposure to COVID-19 and/or a high clinical index of suspicion and patients with bGGO than in the remaining patients (P = 0.00046 and P = 0.0067, respectively). Additionally, no confirmed COVID-19 cases indicating nosocomial spread occurred during the study period. Establishing a comprehensive system that combines rational LAMP-based ICSs with standard ICSs might be useful for preventing nosocomial spread.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/virology , Infection Control/methods , Reverse Transcription/genetics , SARS-CoV-2/genetics , Adult , Clinical Laboratory Techniques/methods , Female , Hospitals , Humans , Male , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , Sensitivity and Specificity , Tokyo , Young Adult
19.
Virol J ; 18(1): 178, 2021 08 30.
Article in English | MEDLINE | ID: covidwho-1379795

ABSTRACT

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, has infected more than 179 million people worldwide. Testing of infected individuals is crucial for identification and isolation, thereby preventing further spread of the disease. Presently, Taqman™ Reverse Transcription Real Time PCR is considered gold standard, and is the most common technique used for molecular testing of COVID-19, though it requires sophisticated equipments, expertise and is also relatively expensive. OBJECTIVE: Development and optimization of an alternate molecular testing method for the diagnosis of COVID-19, through a two step Reverse Transcription Loop-mediated isothermal AMPlification (RT-LAMP). RESULTS: Primers for LAMP were carefully designed for discrimination from other closely related human pathogenic coronaviruses. Care was also taken that primer binding sites are present in conserved regions of SARS-CoV2. Our analysis shows that the primer binding sites are well conserved in all the variants of concern (VOC) and variants of interest (VOI), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427/B.1.429, P.2, B.1.525, P.3, B.1.526 and B.1.617.1. Various DNA polymerases with strand displacement activity were evaluated and conditions were optimized for LAMP amplification and visualization. Different LAMP primer sets were also evaluated using synthetic templates as well as patient samples. CONCLUSION: In a double blind study, the RT-LAMP assay was validated on more than 150 patient samples at two different sites. The RT-LAMP assay appeared to be 89.2% accurate when compared to the Taqman™ rt-RT-PCR assay.


Subject(s)
COVID-19 Testing/methods , COVID-19/virology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/diagnosis , Humans , Reverse Transcription , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Sensitivity and Specificity
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