Ferrobotic swarms enable accessible and adaptable automated viral testing.

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.

End-to-end system for rapid and sensitive early-detection of SARS-CoV-2 for resource-poor and field-test environments using a $51 lab-in-a-backpack.

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.

Rapid molecular diagnosis of Parechovirus infection using the reverse transcription loop-mediated isothermal amplification technique.

OBJECTIVES: Human parechovirus (HPeV), especially HPeV A3 (HPeV3), causes sepsis-like diseases and sudden infant death syndrome in neonates and young infants. Development of rapid and easier diagnostic laboratory tests for HPeVs is desired. METHODS: Original inner primers, outer primers, and loop-primers were designed on the 5' untranslated region of HPeV3. HPeV3 ribonucleic acids (RNAs), other viral RNAs, and clinical stool samples were used to confirm whether the designed primers would allow the detection of HPeV3 with the reverse transcription loop-mediated isothermal amplification (RT-LAMP) technique. RESULTS: Three combinations of primers were created and it was confirmed that all primer sets allowed the detection of HPeV3 RNAs. The primer sets had cross-reactivity with HPeV type 1 (HPeV1), but all sets showed negative results when applied to coxsackievirus, echovirus, enterovirus, norovirus, and adenovirus genomes. Four of six stool samples, obtained from newborn and infant patients with sepsis-like symptoms, showed positive results with our RT-LAMP technique. CONCLUSIONS: This manuscript is the first description of an RT-LAMP for the diagnosis of HPeVs, allowing a faster, easier, and cheaper diagnosis. This technique is clinically useful for newborns and infants who have sepsis-like symptoms.

In-house reverse transcriptase polymerase chain reaction for detection of SARS-CoV-2 with increased sensitivity.

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.

CRISPR-based diagnostics.

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.

An ultra-portable, self-contained point-of-care nucleic acid amplification test for diagnosis of active COVID-19 infection.

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.

EasyNAT MTC assay: A simple, rapid, and low-cost cross-priming amplification method for the detection of mycobacterium tuberculosis suitable for point-of-care testing.

More sensitive, rapid, and affordable diagnostic tools for pulmonary tuberculosis (PTB) are urgently needed. This study aimed to assess the performance of EasyNAT MTC (abbreviation: EasyNAT) (Ustar Biotechnologies, China), a novel isothermal amplification method with a turnaround time of less than two hours that requires a few manual steps to process the sputum. Sputum samples from 249 patients with suspected PTB were subjected to smear, culture, Xpert MTB/RIF (Cepheid, USA) and EasyNAT assay testing. Of the 169 PTB patients, EasyNAT detected more PTB patients than Xpert (72.19% vs. 61.54%, P < 0.05, χ2 = 4.326). Both the Xpert assay and EasyNAT assay detected almost all the culture-positive sputa successfully, but EasyNAT yielded more positive results among the smear-negative and culture-negative PTB cases (44.59% (33/74) vs. 22.97% (17/74), P < 0.01, χ2 = 7.732). Although the specificity of EasyNAT was lower in contrast to Xpert [95.00% (76/80) vs. 98.75% (79/80)], the difference was not significant (P = 0.363, χ2 = 0.826). EasyNAT could be used as an initial test for PTB diagnosis due to its simplicity, rapid turnaround time, high sensitivity, and low cost.

A cost of illness comparison for toxigenic Clostridioides difficile diagnosis algorithms in developing countries.

BACKGROUND: Availability of several commercial tests with different Clostridioides difficile targets contributes to uncertainty and controversies around the optimal diagnostic algorithm. While numerous studies have estimated the financial impact of C. difficile infection, models to guide testing strategies decisions in developing countries, where economic value significantly impacts clinical practice, are currently not available. AIM: To determine the cost of illness of different C. difficile infection (CDI) diagnostic strategies in developing countries. METHODS: Cost-comparison analysis was performed to compare eleven different algorithms of CDI diagnosis. The basis of calculation was a hypothetical cohort of 1000 adult inpatients suspected of CDI. We analyzed turnaround time of test results (i.e., time from taking sample to results emission), test performance (i.e., sensitivity and specificity) and testing costs. Patients were divided in true positive, false positive, true negative and false negative in order to integrate test performance and economics effects. Additional medical costs were calculated: costs of hygiene, medication, length of stay and intensive care unit costs, based on a Brazilian University Hospital costs. CDI prevalence was considered 22.64%. FINDINGS: From laboratory-assisted tests, simultaneous glutamate dehydrogenase (GDH) and toxin A/B rapid immunoassay arbitrated by nucleic acid amplification test (NAAT) presented the lowest cost of illness (450,038.70 USD), whereas standalone NAAT had the highest (523,709.55 USD). Empirical diagnosis only presented the highest overall cost (809,605.44 USD). CONCLUSION: The two-step algorithm with simultaneous GDH and toxin A/B rapid immunoassay arbitrated by NAAT seems to be the best strategy for CDI diagnosis in developing countries.

Rapid Multiplex Loop-Mediated Isothermal Amplification (m-LAMP) Assay for Differential Diagnosis of Leprosy and Post-Kala-Azar Dermal Leishmaniasis.

Leprosy and post-kala-azar dermal leishmaniasis (PKDL) are co-endemic neglected tropical diseases often misdiagnosed because of close resemblance in their clinical manifestations. The test that aids in differential diagnosis of leprosy and PKDL would be useful in endemic areas. Here, we report development of a multiplex loop-mediated isothermal amplification (m-LAMP) assay for differential detection of Mycobacterium leprae and Leishmania donovani using a real-time fluorometer. The m-LAMP assay was rapid with a mean amplification time of 15 minutes, and analytical sensitivity of 1 fg for L. donovani and 100 fg for M. leprae. The distinct mean Tm values for M. leprae and L. donovani allowed differentiation of the two organisms in the m-LAMP assay. Diagnostic sensitivity of the assay was evaluated by using confirmed cases of leprosy (n = 40) and PKDL (n = 40) (tissue and slit aspirate samples). All the leprosy and PKDL samples used in this study were positive by organism-specific QPCR and loop-mediated isothermal amplification assays. The diagnostic sensitivity of the m-LAMP assay was 100% (95% CI: 91.2-100.0%) for detecting PKDL and 95% for leprosy (95% CI: 83.1-99.4%). Our m-LAMP assay was successfully used to detect both M. leprae and L. donovani in a patient coinfected with leprosy and macular PKDL. The m-LAMP assay is rapid, accurate, and applicable for differential diagnosis of leprosy versus PKDL, especially in endemic areas.

Development of a two-step nucleic acid amplification test for accurate diagnosis of the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) remains one of the top 10 leading causes of death globally. The early diagnosis of MTBC can reduce mortality and mitigate disease transmission. However, current nucleic acid amplification diagnostic test methods are generally time-consuming and show suboptimal diagnostic performance, especially in extrapulmonary MTBC samples or acid-fast stain (AFS)-negative cases. Thus, development of an accurate assay for the diagnosis of MTBC is necessary, particularly under the above mentioned conditions. In this study, a single-tube nested real-time PCR assay (N-RTP) was developed and compared with a newly in-house-developed high-sensitivity real-time PCR assay (HS-RTP) using 134 clinical specimens (including 73 pulmonary and 61 extrapulmonary specimens). The amplification efficiency of HS-RTP and N-RTP was 99.8% and 100.7%, respectively. The sensitivity and specificity of HS-RTP and N-RTP for the diagnosis of MTBC in these specimens were 97.5% (77/79) versus 94.9% (75/79) and 80.0% (44/55) versus 89.1% (49/55), respectively. The sensitivity and specificity of HS-RTP and N-RTP for the diagnosis of MTBC in pulmonary specimens were 96.3% (52/54) versus 96.3% (52/54) and 73.7.0% (14/19) versus 89.5% (17/19), respectively; in extrapulmonary specimens, the sensitivity and specificity of HS-RTP and N-RTP were 100% (25/25) versus 92% (23/25) and 83.3% (30/36) versus 88.9% (32/36), respectively. Among the AFS-negative cases, the sensitivity and specificity of HS-RTP and N-RTP were 97.0% (32/33) versus 90.9% (30/33) and 88.0% (44/50) versus 92.0% (46/50), respectively. Overall, the sensitivity of HS-RTP was higher than that of N-RTP, and the performance was not compromised in extrapulmonary specimens and under AFS-negative conditions. In contrast, the specificity of the N-RTP assay was higher than that of the HS-RTP assay in all types of specimens. In conclusion, the HS-RTP assay would be useful for screening patients suspected of exhibiting an MTBC infection due to its higher sensitivity, while the N-RTP assay could be used for confirmation because of its higher specificity. Our results provide a two-step method (screen to confirm) that simultaneously achieves high sensitivity and specificity in the diagnosis of MTBC.

Emulsion-based isothermal nucleic acid amplification for rapid SARS-CoV-2 detection via angle-dependent light scatter analysis.

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.

Effectiveness and cost-effectiveness of four different strategies for SARS-CoV-2 surveillance in the general population (CoV-Surv Study): a structured summary of a study protocol for a cluster-randomised, two-factorial controlled trial.

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.

Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

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.

A rapid diagnosis of SARS-CoV-2 using DNA hydrogel formation on microfluidic pores.

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.

Loop-mediated isothermal amplification (LAMP): An effective molecular point-of-care technique for the rapid diagnosis of coronavirus SARS-CoV-2.

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.

Point-of-care diagnostic tests for influenza in the emergency department: A cost-effectiveness analysis in a high-risk population from a Canadian perspective.

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.

Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus.

Tristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbreaks. Currently, the standard molecular techniques for CTV detection include RT-PCR and RT-qPCR. These diagnostic methods are highly sensitive but time consuming, labor intensive and require sophisticated expensive instruments, thus not suitable for point-of-care use. In the present study, we report the development of a rapid, sensitive, robust, reliable, and highly specific reverse transcription-RPA technique coupled with a lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA). RT-RPA technique was standardized to amplify the coat protein gene of CTV (CTV-p25) and detect double labeled amplicons on a sandwich immunoassay by designing specific labeled primer pair and probe combinations. The optimally performing primer set (CTRPA-F1/CTRPA-R9-Btn) and the corresponding TwistAmp nfo probe (CTRPA-Probe) was optimized for temperature and reaction time using purified cDNA and viral RNA as template. The sensitivity of the developed assay was compared with other detection techniques using in vitro-transcribed RNA. The efficacy and specificity of the assay was evaluated using CTV positive controls, healthy samples, field grown citrus plants of unknown status, and other virus and bacterial pathogens that infect citrus plants. The RT-RPA-LFICA was able to detect ≤ 141 fg of RNA when cDNA used as a template. The assay detected ≤ 0.23 ng/µl of CTV RNA when directly used as template without cross-reactivity with other citrus pathogens. Best results were achieved at the isothermal temperature of 40 °C within 15-20 min. The study demonstrated that RT-RPA-LFICA has potential to become an improved detection technique for end users in bud-wood certification and quarantine programs and a promising platform for rapid point-of-care diagnostics for citrus farmers and small nurseries in low resource settings.

Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks.

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s.

Detecting SARS-CoV-2 at point of care: preliminary data comparing loop-mediated isothermal amplification (LAMP) to polymerase chain reaction (PCR).

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.