Can a field molecular diagnosis be accurate? A performance evaluation of colorimetric RT-LAMP for the detection of SARS-CoV-2 in a hospital setting.
TL;DR: In this article, a colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) was used to detect SARS-CoV-2 using different protocols.
Abstract: SARS-CoV-2 currently represents a serious global public health problem. Non-pharmaceutical intervention measures (NPIs) have been widely adopted, and the testing strategy since the beginning of the infection is the most effective tool for tracking, isolating, and minimizing transmission. The high operating costs and the need for sophisticated instrumentation related to gold standard diagnostic for COVID-19, Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR), have highlighted the urgency and importance of developing and applying new diagnostic techniques, especially in places with scarce resources. Thus, alternative molecular tests, such as Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP), based on isothermal amplification have been used to detect SARS-CoV-2 using different protocols. The potential for field application of RT-LAMP is due to the lower cost and time and not requiring high-cost instrumentation. Here, we evaluate the colorimetric RT-LAMP to detect SARS-CoV-2 in a hospital environment and correlate its performance with tests performed in a reference laboratory. The analysis performed at the hospital showed high sensitivity (88.89%), specificity (98.55%), accuracy (95.83%), and a Cohen's kappa of 0.895. However, we achieved 100% of agreement when comparing the RT-LAMP results with the gold standard (qRT-PCR) results for samples with Ct < 30 in the hospital-based test. In addition, a similar performance was found in the field compared to the reference laboratory, corroborating the proposal to apply the test directly at point-of-care.
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TL;DR: In this paper , the FastProof 30 min-TTR SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP) method using leftover RNA samples extracted from 315 nasopharyngeal swabs.
13 citations
TL;DR: This review aims to compile the state-of-the-art in the field of nucleic acid amplification tests (NAATs) used for SARS-CoV-2 detection, either at the clinic level, or at the Point-Of-Care (POC), thus focusing on isothermal and non-isothermal amplification-based diagnostics.
Abstract: COVID-19 pandemic ignited the development of countless molecular methods for the diagnosis of SARS-CoV-2 based either on nucleic acid, or protein analysis, with the first establishing as the most used for routine diagnosis. The methods trusted for day to day analysis of nucleic acids rely on amplification, in order to enable specific SARS-CoV-2 RNA detection. This review aims to compile the state-of-the-art in the field of nucleic acid amplification tests (NAATs) used for SARS-CoV-2 detection, either at the clinic level, or at the Point-Of-Care (POC), thus focusing on isothermal and non-isothermal amplification-based diagnostics, while looking carefully at the concerning virology aspects, steps and instruments a test can involve. Following a theme contextualization in introduction, topics about fundamental knowledge on underlying virology aspects, collection and processing of clinical samples pave the way for a detailed assessment of the amplification and detection technologies. In order to address such themes, nucleic acid amplification methods, the different types of molecular reactions used for DNA detection, as well as the instruments requested for executing such routes of analysis are discussed in the subsequent sections. The benchmark of paradigmatic commercial tests further contributes toward discussion, building on technical aspects addressed in the previous sections and other additional information supplied in that part. The last lines are reserved for looking ahead to the future of NAATs and its importance in tackling this pandemic and other identical upcoming challenges.
12 citations
TL;DR: In this article, a review of the most recent diagnostic approaches and their potential in the context of the COVID-19 pandemic has been presented, highlighting the potential of each research direction.
Abstract: COVID-19 is one of the most severe global health crises that humanity has ever faced. Researchers have restlessly focused on developing solutions for monitoring and tracing the viral culprit, SARS-CoV-2, as vital steps to break the chain of infection. Even though biomedical engineering (BME) is considered a rising field of medical sciences, it has demonstrated its pivotal role in nurturing the maturation of COVID-19 diagnostic technologies. Within a very short period of time, BME research applied to COVID-19 diagnosis has advanced with ever-increasing knowledge and inventions, especially in adapting available virus detection technologies into clinical practice and exploiting the power of interdisciplinary research to design novel diagnostic tools or improve the detection efficiency. To assist the development of BME in COVID-19 diagnosis, this review highlights the most recent diagnostic approaches and evaluates the potential of each research direction in the context of the pandemic.
8 citations
TL;DR: In this paper , the authors presented an RT-LAMP-based method for detecting SARS-CoV-2 lineages, which can be used as a screening approach for variants in countries with poor sequencing coverage.
Abstract: Despite the advance of vaccination worldwide, epidemic waves caused by more transmissible and immune evasive genetic variants of SARS-CoV-2 have sustained the ongoing pandemic of COVID-19. Monitoring such variants is expensive, as it usually relies on whole-genome sequencing methods. Therefore, it is necessary to develop alternatives that could help identify samples from specific variants. Reverse transcription loop-mediated isothermal amplification is a method that has been increasingly used for nucleic acid amplification, as it is cheaper and easier to perform when compared to other molecular techniques. As a proof of concept that can help distinguish variants, we present an RT-LAMP assay capable of detecting samples carrying a group of mutations that can be related to specific SARS-CoV-2 lineages, here demonstrated for the Variant of Concern Gamma. We tested 60 SARS-CoV-2 RNA samples extracted from swab samples and the reaction showed a sensitivity of 93.33%, a specificity of 88.89% and a kappa value of 0.822 for samples with a Ct ≤ 22.93. The RT-LAMP assay demonstrated to be useful to distinguish VOC Gamma and may be of particular interest as a screening approach for variants in countries with poor sequencing coverage.
2 citations
TL;DR: R reverse transcription loop-mediated isothermal amplification (RT-LAMP) is proposed to be an alternative to help monitor variants, especially in countries with scarce resources.
Abstract: Two lineages (BA.1 and BA.2) of the Omicron variant are the main ones responsible for the recent COVID-19 pandemic waves worldwide. Monitoring the prevalence and spread of these variants is important as the presence of mutations might lower the efficacy of vaccines and hinder the benefits of monoclonal antibody therapies. Although the need to screen these new lineages is emerging, genetic sequencing is scarce due to its high cost. Alternatively, we propose using reverse transcription loop-mediated isothermal amplification (RT-LAMP) to infer the prevalence of these lineages and aid in genomic surveillance in countries with limited genetic sequencing capacity. For this, we designed specific primers and tested them on a panel of 267 sequenced RNA genomes from different lineages. The test for BA.1 and its descendants showed 96.63% sensitivity, 100% specificity, and 98.85% accuracy, and the test for BA.2 and descendants showed 90.00% sensitivity, 98.85% specificity, and 98.52% accuracy. These results demonstrate the potential of RT-LAMP to be an alternative to help monitor variants, especially in countries with scarce resources.
1 citations
References
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TL;DR: The epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of patients with laboratory-confirmed 2019-nCoV infection in Wuhan, China, were reported.
36,578 citations
TL;DR: Characteristics of patients who died were in line with the MuLBSTA score, an early warning model for predicting mortality in viral pneumonia, and further investigation is needed to explore the applicability of the Mu LBSTA scores in predicting the risk of mortality in 2019-nCoV infection.
16,282 citations
TL;DR: A validated diagnostic workflow for 2019-nCoV is presented, its design relying on close genetic relatedness of 2019- nCoV with SARS coronavirus, making use of synthetic nucleic acid technology.
Abstract: Background The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. Aim We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Methods Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. Results The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive – Global (EVAg), a European Union infrastructure project. Conclusion The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
6,229 citations
TL;DR: It is estimated that 86% of all infections were undocumented before the 23 January 2020 travel restrictions, which explains the rapid geographic spread of SARS-CoV-2 and indicates that containment of this virus will be particularly challenging.
Abstract: Estimation of the prevalence and contagiousness of undocumented novel coronavirus [severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2)] infections is critical for understanding the overall prevalence and pandemic potential of this disease. Here, we use observations of reported infection within China, in conjunction with mobility data, a networked dynamic metapopulation model, and Bayesian inference, to infer critical epidemiological characteristics associated with SARS-CoV-2, including the fraction of undocumented infections and their contagiousness. We estimate that 86% of all infections were undocumented [95% credible interval (CI): 82–90%] before the 23 January 2020 travel restrictions. The transmission rate of undocumented infections per person was 55% the transmission rate of documented infections (95% CI: 46–62%), yet, because of their greater numbers, undocumented infections were the source of 79% of the documented cases. These findings explain the rapid geographic spread of SARS-CoV-2 and indicate that containment of this virus will be particularly challenging.
3,324 citations
TL;DR: An improved simple visual detection system for the results of the LAMP reaction that enables visual discrimination of results without costly specialized equipment should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.
Abstract: As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Critical to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amount of DNA is synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combines with divalent metallic ion to form an insoluble salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction solution allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30-60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.
1,521 citations