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1.
PLoS One ; 17(1): e0259886, 2022.
Article in English | MEDLINE | ID: covidwho-1666744

ABSTRACT

COVID-19 has exposed stark inequalities between resource-rich and resource-poor countries. International UN- and WHO-led efforts, such as COVAX, have provided SARS-CoV-2 vaccines but half of African countries have less than 2% vaccinated in their population, and only 15 have reached 10% by October 2021, further disadvantaging local economic recovery. Key for this implementation and preventing further mutation and spread is the frequency of voluntary [asymptomatic] testing. It is limited by expensive PCR and LAMP tests, uncomfortable probes deep in the throat or nose, and the availability of hardware to administer in remote locations. There is an urgent need for an inexpensive "end-to-end" system to deliver sensitive and reliable, non-invasive tests in resource-poor and field-test conditions. We introduce a non-invasive saliva-based LAMP colorimetric test kit and a $51 lab-in-a-backpack system that detects as few as 4 viral RNA copies per µL. It consists of eight chemicals, a thermometer, a thermos bottle, two micropipettes and a 1000-4000 rcf electronically operated centrifuge made from recycled computer hard drives (CentriDrive). The centrifuge includes a 3D-printed rotor and a 12 V rechargeable Li-ion battery, and its 12 V standard also allows wiring directly to automobile batteries, to enable field-use of this and other tests in low infrastructure settings. The test takes 90 minutes to process 6 samples and has reagent costs of $3.5 per sample. The non-invasive nature of saliva testing would allow higher penetration of testing and wider adoption of the test across cultures and settings (including refugee camps and disaster zones). The attached graphical procedure would make the test suitable for self-testing at home, performing it in the field, or in mobile testing centers by minimally trained staff.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , COVID-19/virology , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/methods , Colorimetry , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Systems , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Saliva/virology
2.
Rev Med Virol ; 31(6): e2215, 2021 11.
Article in English | MEDLINE | ID: covidwho-1573992

ABSTRACT

The novel coronavirus disease-2019 (Covid-19) public health emergency has caused enormous loss around the world. This pandemic is a concrete example of the existing gap between availability of advanced diagnostics and current need for cost-effective methodology. The advent of the loop-mediated isothermal amplification (LAMP) assay provided an innovative tool for establishing a rapid diagnostic technique based on the molecular amplification of pathogen RNA or DNA. In this review, we explore the applications, diagnostic effectiveness of LAMP test for molecular diagnosis and surveillance of severe acute respiratory syndrome coronavirus 2. Our results show that LAMP can be considered as an effective point-of-care test for the diagnosis of Covid-19 in endemic areas, especially for low- and middle-income countries.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Point-of-Care Testing/organization & administration , SARS-CoV-2/genetics , Bibliometrics , COVID-19/epidemiology , COVID-19/pathology , COVID-19/virology , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/instrumentation , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Testing/economics , RNA, Viral/genetics , SARS-CoV-2/pathogenicity , Sensitivity and Specificity
3.
Trials ; 22(1): 39, 2021 Jan 08.
Article in English | MEDLINE | ID: covidwho-1440947

ABSTRACT

OBJECTIVES: In this cluster-randomised controlled study (CoV-Surv Study), four different "active" SARS-CoV-2 testing strategies for general population surveillance are evaluated for their effectiveness in determining and predicting the prevalence of SARS-CoV-2 infections in a given population. In addition, the costs and cost-effectiveness of the four surveillance strategies will be assessed. Further, this trial is supplemented by a qualitative component to determine the acceptability of each strategy. Findings will inform the choice of the most effective, acceptable and affordable strategy for SARS-CoV-2 surveillance, with the most effective and cost-effective strategy becoming part of the local public health department's current routine health surveillance activities. Investigating its everyday performance will allow us to examine the strategy's applicability to real time prevalence prediction and the usefulness of the resulting information for local policy makers to implement countermeasures that effectively prevent future nationwide lockdowns. The authors would like to emphasize the importance and relevance of this study and its expected findings in the context of population-based disease surveillance, especially in respect to the current SARS-CoV-2 pandemic. In Germany, but also in many other countries, COVID-19 surveillance has so far largely relied on passive surveillance strategies that identify individuals with clinical symptoms, monitor those cases who then tested positive for the virus, followed by tracing of individuals in close contact to those positive cases. To achieve higher effectiveness in population surveillance and to reliably predict the course of an outbreak, screening and monitoring of infected individuals without major symptoms (about 40% of the population) will be necessary. While current testing capacities are also used to identify such asymptomatic cases, this rather passive approach is not suitable in generating reliable population-based estimates of the prevalence of asymptomatic carriers to allow any dependable predictions on the course of the pandemic. To better control and manage the SARS-CoV-2 pandemic, current strategies therefore need to be complemented by an active surveillance of the wider population, i.e. routinely conducted testing and monitoring activities to identify and isolate infected individuals regardless of their clinical symptoms. Such active surveillance strategies will enable more effective prevention of the spread of the virus as they can generate more precise population-based parameters during a pandemic. This essential information will be required in order to determine the best strategic and targeted short-term countermeasures to limit infection spread locally. TRIAL DESIGN: This trial implements a cluster-randomised, two-factorial controlled, prospective, interventional, single-blinded design with four study arms, each representing a different SARS-CoV-2 testing and surveillance strategy. PARTICIPANTS: Eligible are individuals age 7 years or older living in Germany's Rhein-Neckar Region who consent to provide a saliva sample (all four arms) after completion of a brief questionnaire (two arms only). For the qualitative component, different samples of study participants and non-participants (i.e. eligible for study, but refuse to participate) will be identified for additional interviews. For these interviews, only individuals age 18 years or older are eligible. INTERVENTION AND COMPARATOR: Of the four surveillance strategies to be assessed and compared, Strategy A1 is considered the gold standard for prevalence estimation and used to determine bias in other arms. To determine the cost-effectiveness, each strategy is compared to status quo, defined as the currently practiced passive surveillance approach. Strategy A1: Individuals (one per household) receive information and study material by mail with instructions on how to produce a saliva sample and how to return the sample by mail. Once received by the laboratory, the sample is tested for SARS-CoV-2 using Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP). Strategy A2: Individuals (one per household) receive information and study material by mail with instructions on how to produce their own as well as saliva samples from each household member and how to return these samples by mail. Once received by the laboratory, the samples are tested for SARS-CoV-2 using RT-LAMP. Strategy B1: Individuals (one per household) receive information by mail on how to complete a brief pre-screening questionnaire which asks about COVID-19 related clinical symptoms and risk exposures. Only individuals whose pre-screening score crosses a defined threshold, will then receive additional study material by mail with instructions on how to produce a saliva sample and how to return the sample by mail. Once received by the laboratory, the saliva sample is tested for SARS-CoV-2 using RT-LAMP. Strategy B2: Individuals (one per household) receive information by mail on how to complete a brief pre-screening questionnaire which asks about COVID-19 related clinical symptoms. Only individuals whose pre-screening score crosses a defined threshold, will then receive additional study material by mail with instructions how to produce their own as well as saliva samples from each household member and how to return these samples by mail. Once received by the laboratory, the samples are tested for SARS-CoV-2 using RT-LAMP. In each strategy, RT-LAMP positive samples are additionally analyzed with qPCR in order to minimize the number of false positives. MAIN OUTCOMES: The identification of the one best strategy will be determined by a set of parameters. Primary outcomes include costs per correctly screened person, costs per positive case, positive detection rate, and precision of positive detection rate. Secondary outcomes include participation rate, costs per asymptomatic case, prevalence estimates, number of asymptomatic cases per study arm, ratio of symptomatic to asymptomatic cases per study arm, participant satisfaction. Additional study components (not part of the trial) include cost effectiveness of each of the four surveillance strategies compared to passive monitoring (i.e. status quo), development of a prognostic model to predict hospital utilization caused by SARS-CoV-2, time from test shipment to test application and time from test shipment to test result, and perception and preferences of the persons to be tested with regard to test strategies. RANDOMISATION: Samples are drawn in three batches of three continuous weeks. Randomisation follows a two-stage process. First, a total of 220 sampling points have been allocated to the three different batches. To obtain an integer solution, the Cox-algorithm for controlled rounding has been used. Afterwards, sample points have been drawn separately per batch, following a probability proportional to size (PPS) random sample. Second, for each cluster the same number of residential addresses is randomly sampled from the municipal registries (self-weighted sample of individuals). The 28,125 addresses drawn per municipality are then randomly allocated to the four study arms A1, A2, B1, and B2 in the ratio 5 to 2.5 to 14 to 7 based on the expected response rates in each arm and the sensitivity and specificity of the pre-screening tool as applied in strategy B1 and B2. Based on the assumptions, this allocation should yield 2500 saliva samples in each strategy. Although a municipality can be sampled by multiple batches and the overall number of addresses per municipality might vary, the number of addresses contacted in each arm is kept constant. BLINDING (MASKING): The design is single-blinded, meaning the staff conducting the SARS-CoV-2 tests are unaware of the study arm assignment of each single participant and test sample. SAMPLE SIZES: Total sample size for the trial is 10,000 saliva samples equally allocated to the four study arms (i.e. 2,500 participants per arm). For the qualitative component, up to 60 in-depth interviews will be conducted with about 30 study participants (up to 15 in each arm A and B) and 30 participation refusers (up to 15 in each arm A and B) purposefully selected from the quantitative study sample to represent a variety of gender and ages to explore experiences with admission or rejection of study participation. Up to 25 asymptomatic SARS-CoV-2 positive study participants will be purposefully selected to explore the way in which asymptomatic men and women diagnosed with SARS-CoV-2 give meaning to their diagnosis and to the dialectic between feeling concurrently healthy and yet also being at risk for transmitting COVID-19. In addition, 100 randomly selected study participants will be included to explore participants' perspective on testing processes and implementation. TRIAL STATUS: Final protocol version is "Surveillance_Studienprotokoll_03Nov2020_v1_2" from November 3, 2020. Recruitment started November 18, 2020 and is expected to end by or before December 31, 2020. TRIAL REGISTRATION: The trial is currently being registered with the German Clinical Trials Register (Deutsches Register Klinischer Studien), DRKS00023271 ( https://www.drks.de/drks_web/navigate.do?navigationId=trial . HTML&TRIAL_ID=DRKS00023271). Retrospectively registered 30 November 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.


Subject(s)
COVID-19 Nucleic Acid Testing/economics , COVID-19/diagnosis , COVID-19/economics , Health Care Costs , Molecular Diagnostic Techniques/economics , Nucleic Acid Amplification Techniques/economics , SARS-CoV-2/genetics , Saliva/virology , Surveys and Questionnaires/economics , COVID-19/epidemiology , COVID-19/virology , Cost-Benefit Analysis , Female , Germany/epidemiology , Humans , Male , Population Surveillance , Predictive Value of Tests , Prevalence , Randomized Controlled Trials as Topic , Reproducibility of Results , Single-Blind Method
4.
Sci Rep ; 11(1): 17878, 2021 09 09.
Article in English | MEDLINE | ID: covidwho-1402125

ABSTRACT

As the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Humans , Nucleic Acid Amplification Techniques/economics , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , Reverse Transcriptase Polymerase Chain Reaction/economics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Viroporin Proteins/genetics
5.
Sci Rep ; 11(1): 15176, 2021 07 26.
Article in English | MEDLINE | ID: covidwho-1327219

ABSTRACT

There is currently a high level of demand for rapid COVID-19 tests, that can detect the onset of the disease at point of care settings. We have developed an ultra-portable, self-contained, point-of-care nucleic acid amplification test for diagnosis of active COVID-19 infection, based on the principle of loop mediated isothermal amplification (LAMP). The LAMP assay is 100% sensitive and specific to detect a minimum of 300 RNA copies/reaction of SARS-CoV-2. All of the required sample transportation, lysing and amplification steps are performed in a standalone disposable cartridge, which is controlled by a battery operated, pocket size (6x9x4cm3) unit. The test is easy to operate and does not require skilled personnel. The total time from sample to answer is approximately 35 min; a colorimetric readout indicates positive or negative results. This portable diagnostic platform has significant potential for rapid and effective testing in community settings. This will accelerate clinical decision making, in terms of effective triage and timely therapeutic and infection control interventions.


Subject(s)
COVID-19 Nucleic Acid Testing/instrumentation , COVID-19/diagnosis , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Testing , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/economics , Equipment Design , Humans , Molecular Diagnostic Techniques/economics , Nucleic Acid Amplification Techniques/economics , Point-of-Care Testing/economics , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Time Factors
6.
Nat Biomed Eng ; 5(7): 643-656, 2021 07.
Article in English | MEDLINE | ID: covidwho-1324420

ABSTRACT

The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiological surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of molecular targets relevant to human health.


Subject(s)
CRISPR-Cas Systems/genetics , Communicable Diseases/diagnosis , Nucleic Acid Amplification Techniques/methods , Nucleic Acids/analysis , Communicable Diseases/microbiology , Communicable Diseases/virology , Genetic Diseases, Inborn/diagnosis , Humans , Nucleic Acid Amplification Techniques/economics , Nucleic Acids/metabolism , Point-of-Care Systems , Polymorphism, Single Nucleotide , Sequence Analysis, DNA
7.
Biosens Bioelectron ; 179: 113099, 2021 May 01.
Article in English | MEDLINE | ID: covidwho-1086796

ABSTRACT

The SARS-CoV-2 pandemic, an ongoing global health crisis, has revealed the need for new technologies that integrate the sensitivity and specificity of RT-PCR tests with a faster time-to-detection. Here, an emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to allow for the compartmentalization of LAMP reactions, leading to faster changes in emulsion characteristics, and thus lowering time-to-detection. Within these droplets, ongoing LAMP reactions lead to adsorption of amplicons to the water-oil interface, causing a decrease in interfacial tension, resulting in smaller emulsion diameters. Changes in emulsion diameter allow for the monitoring of the reaction by use of angle-dependent light scatter (based off Mie scatter theory). Mie scatter simulations confirmed that light scatter intensity is diameter-dependent and smaller colloids have lower intensity values compared to larger colloids. Via spectrophotometers and fiber optic cables placed at 30° and 60°, light scatter intensity was monitored. Scatter intensities collected at 5 min, 30° could statistically differentiate 10, 103, and 105 copies/µL initial concentrations compared to NTC. Similarly, 5 min scatter intensities collected at 60° could statistically differentiate 105 copies/µL initial concentrations in comparison to NTC. The use of both angles during the eLAMP assay allows for distinction between high and low initial target concentrations. The efficacy of a smartphone-based platform was also tested and had a similar limit of detection and assay time of less than 10 min. Furthermore, fluorescence-labeled primers were used to validate target nucleic acid amplification. Compared to existing LAMP assays for SARS-CoV-2 detection, these times-to-detections are very rapid.


Subject(s)
COVID-19 Nucleic Acid Testing/instrumentation , COVID-19/diagnosis , Dynamic Light Scattering/instrumentation , Emulsions/chemistry , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , SARS-CoV-2/isolation & purification , Biosensing Techniques/economics , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/methods , Dynamic Light Scattering/economics , Dynamic Light Scattering/methods , Equipment Design , Humans , Limit of Detection , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/methods , Smartphone , Time Factors
8.
Biosens Bioelectron ; 177: 113005, 2021 Apr 01.
Article in English | MEDLINE | ID: covidwho-1033431

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has been a major public health challenge in 2020. Early diagnosis of COVID-19 is the most effective method to control disease spread and prevent further mortality. As such, a high-precision and rapid yet economic assay method is urgently required. Herein, we propose an innovative method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using isothermal amplification of nucleic acids on a mesh containing multiple microfluidic pores. Hybridization of pathogen DNA and immobilized probes forms a DNA hydrogel by rolling circle amplification and, consequently, blocks the pores to prevent fluid movement, as observed. Following optimization of several factors, including pore size, mesh location, and precision microfluidics, the limit of detection (LOD) for SARS-CoV-2 was determined to be 0.7 aM at 15-min incubation. These results indicate rapid, easy, and effective detection with a moderate-sized LOD of the target pathogen by remote point-of-care testing and without the requirement of any sophisticated device.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Hydrogels/chemistry , Immobilized Nucleic Acids/chemistry , Point-of-Care Testing , SARS-CoV-2/isolation & purification , Biosensing Techniques/economics , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , COVID-19/virology , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/instrumentation , DNA Probes/chemistry , DNA Probes/genetics , Equipment Design , Humans , Immobilized Nucleic Acids/genetics , Lab-On-A-Chip Devices , Limit of Detection , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/instrumentation , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics
9.
PLoS One ; 16(1): e0243712, 2021.
Article in English | MEDLINE | ID: covidwho-1024413

ABSTRACT

To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT-PCR (Reverse transcription-Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT-LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross-reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called "COVIDISC" to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.


Subject(s)
COVID-19 Testing/instrumentation , COVID-19/diagnosis , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Testing , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19 Testing/economics , Equipment Design , Humans , Limit of Detection , Molecular Diagnostic Techniques/economics , Nucleic Acid Amplification Techniques/economics , Point-of-Care Testing/economics , RNA, Viral/isolation & purification , SARS-CoV-2/isolation & purification , Time Factors
10.
PLoS One ; 15(11): e0242255, 2020.
Article in English | MEDLINE | ID: covidwho-949088

ABSTRACT

BACKGROUND: Our objective was to assess the cost-effectiveness of novel rapid diagnostic tests: rapid influenza diagnostic tests (RIDT), digital immunoassays (DIA), rapid nucleic acid amplification tests (NAAT), and other treatment algorithms for influenza in high-risk patients presenting to hospital with influenza-like illness (ILI). METHODS: We developed a decision-analytic model to assess the cost-effectiveness of diagnostic test strategies (RIDT, DIA, NAAT, clinical judgement, batch polymerase chain reaction) preceding treatment; no diagnostic testing and treating everyone; and not treating anyone. We modeled high-risk 65-year old patients from a health payer perspective and accrued outcomes over a patient's lifetime. We reported health outcomes, quality-adjusted life years (QALYs), healthcare costs, and net health benefit (NHB) to measure cost-effectiveness per cohort of 100,000 patients. RESULTS: Treating everyone with no prior testing was the most cost-effective strategy, at a cost-effectiveness threshold of $50,000/QALY, in over 85% of simulations. This strategy yielded the highest NHB of 15.0344 QALYs, but inappropriately treats all patients without influenza. Of the novel rapid diagnostics, NAAT resulted in the highest NHB (15.0277 QALYs), and the least number of deaths (1,571 per 100,000). Sensitivity analyses determined that results were most impacted by the pretest probability of ILI being influenza, diagnostic test sensitivity, and treatment effectiveness. CONCLUSIONS: Based on our model, treating high-risk patients presenting to hospital with influenza-like illness, without performing a novel rapid diagnostic test, resulted in the highest NHB and was most cost-effective. However, consideration of whether treatment is appropriate in the absence of diagnostic confirmation should be taken into account for decision-making by clinicians and policymakers.


Subject(s)
Cost-Benefit Analysis , Influenza, Human/diagnosis , Point-of-Care Testing/economics , Aged , Canada , Emergency Service, Hospital/economics , Female , Health Care Costs , Humans , Immunoassay/economics , Influenza, Human/mortality , Influenza, Human/therapy , Male , Nucleic Acid Amplification Techniques/economics , Quality-Adjusted Life Years
11.
BMC Infect Dis ; 20(1): 783, 2020 Oct 20.
Article in English | MEDLINE | ID: covidwho-883564

ABSTRACT

BACKGROUND: A cost effective and efficient diagnostic tool for COVID-19 as near to the point of care (PoC) as possible would be a game changer in the current pandemic. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 min, alongside standard methods in a real-life clinical setting. METHODS: This prospective service improvement project piloted an RT-LAMP method on nasal and pharyngeal swabs on 21 residents of a high dependency care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We recorded vital signs of patients to correlate clinical and laboratory information and calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of a single swab using RT-LAMP compared with the current standard, RT-PCR, as per Standards for Reporting Diagnostic Accuracy Studies (STARD) guidelines. RESULTS: The novel method accurately detected 8/10 RT-PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a "gold standard", the sensitivity and specificity of a single novel test were 80 and 73% respectively. PPV was 73% and NPV was 83%. Incorporating retesting of low signal RT-LAMP positives improved the specificity to 100%. We also speculate that hypothermia may be a significant early clinical sign of COVID-19. CONCLUSIONS: RT-LAMP testing for SARS-CoV-2 was found to be promising, fast and to work equivalently to RT-PCR methods. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the PoC. RT-LAMP could be deployed in mobile community testing units, care homes and hospitals to detect disease early and prevent spread.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Point-of-Care Systems , Polymerase Chain Reaction/methods , Preliminary Data , Aged , Aged, 80 and over , COVID-19 , COVID-19 Testing , Coronavirus Infections/virology , Data Accuracy , Female , Humans , Male , Middle Aged , Molecular Diagnostic Techniques/economics , Nucleic Acid Amplification Techniques/economics , Pandemics , Pneumonia, Viral/virology , Polymerase Chain Reaction/economics , Prospective Studies , SARS-CoV-2 , Sensitivity and Specificity
12.
Proc Natl Acad Sci U S A ; 117(39): 24450-24458, 2020 09 29.
Article in English | MEDLINE | ID: covidwho-752376

ABSTRACT

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Betacoronavirus/genetics , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/economics , Colorimetry , Coronavirus Infections/economics , Coronavirus Infections/virology , Genome, Viral/genetics , Humans , Hydrogen-Ion Concentration , Molecular Diagnostic Techniques/economics , Nucleic Acid Amplification Techniques/economics , Pandemics , Pneumonia, Viral/virology , Polyproteins , RNA Stability , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/isolation & purification , SARS-CoV-2 , Sensitivity and Specificity , Time Factors , Viral Proteins/genetics , Virus Inactivation
13.
PLoS Pathog ; 16(8): e1008705, 2020 08.
Article in English | MEDLINE | ID: covidwho-732988

ABSTRACT

The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.


Subject(s)
Betacoronavirus/genetics , CRISPR-Cas Systems/genetics , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Coronavirus Infections/genetics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/genetics , Bacteria/genetics , COVID-19 , COVID-19 Testing , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genes, Viral/genetics , Genome, Viral/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/methods , Pandemics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2 , Sensitivity and Specificity
14.
PLoS One ; 15(6): e0234682, 2020.
Article in English | MEDLINE | ID: covidwho-595220

ABSTRACT

Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30-45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30-45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Point-of-Care Testing , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/genetics , COVID-19 , Coronavirus Infections/virology , DNA Primers , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/instrumentation , Pandemics , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/economics , Real-Time Polymerase Chain Reaction/instrumentation , SARS-CoV-2 , Sensitivity and Specificity , Time Factors
15.
Med Hypotheses ; 141: 109786, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-116581

ABSTRACT

Recently, a novel coronavirus (SARS-CoV-2; coronavirus disease 2019, COVID-19) has emerged, rapidly spreading and severely straining the capacity of the global health community. Many nations are employing combinations of containment and mitigation strategies, where early diagnosis of COVID-19 is vital in controlling illness progression and limiting viral spread within the population. Thus, rapid and accurate methods of early detection are vital to contain COVID-19 and prevent further spread and predicted subsequent infectious waves of viral recurrence in future. Immediately after its initial characterization, Chinese and American Centers for Disease Control and Prevention (CDCs) rapidly employed molecular assays for detection of COVID-19, mostly employing real-time polymerase chain reaction (RT-PCR) methods. However, such methods require specific expensive items of equipment and highly trained analysts, requiring upwards of 4-8 h to process. These requirements coupled with associated financial pressures may prevent effective deployment of such diagnostic tests. Loop mediated isothermal amplification(LAMP) is method of nucleic acid amplification which exhibits increased sensitivity and specificity are significantly rapid, and do not require expensive reagents or instruments, which aids in cost reduction for coronavirus detection. Studies have shown the successful application of LAMP assays in various forms to detect coronavirus RNA in patient samples, demonstrating that 1-10 copies of viral RNA template per reaction are sufficient for successful detection, ~100-fold more sensitive than conventional RT-PCR methods. Importantly, studies have also now demonstrated the effectiveness of LAMP methodology in the detection of SARS-CoV-2 RNA at significantly low levels, particularly following numerous improvements to LAMP assay protocols. We hypothesise that recent advancements in enhanced LAMP protocols assay perhaps represent the best chance for a rapid and robust assay for field diagnosis of COVID-19, without the requirement of specialized equipment and highly trained professionals to interpret results. Herein, we present our arguments with a view to disseminate such findings, to assist the combat of this virus that is proving so devastating. We hope that this strategy could be applied rapidly, and confirmed for viability with clinical samples, before being rolled out for mass-diagnostic testing in these current times.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Pandemics , Pneumonia, Viral/diagnosis , Betacoronavirus/genetics , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Clinical Laboratory Techniques/economics , Coronavirus Infections/economics , Coronavirus Infections/virology , DNA Primers , Early Diagnosis , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/methods , Pandemics/economics , Pneumonia, Viral/economics , Pneumonia, Viral/virology , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/economics , SARS-CoV-2 , Sensitivity and Specificity , Time Factors
16.
Emerg Microbes Infect ; 9(1): 998-1007, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-88525

ABSTRACT

The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 102 RNA copies close to that of qRT-PCR. Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.


Subject(s)
Coronavirus Infections/diagnosis , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Humans , Limit of Detection , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/standards , Nucleocapsid Proteins/genetics , Pandemics , Phosphoproteins , Pneumonia, Viral/virology , SARS-CoV-2 , Time Factors
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