COVID-19 Tests | Wish List

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Abacus Diagnostica's new multiplex assay provides reliable influenza, COVID-19 diagnosis in 75 minutes

9/30/20 ... nutes.aspx

Abacus Diagnostica is a small Finnish company developing and manufacturing assays and devices for rapid molecular diagnostics. Since the beginning of COVID-19 outbreak the company has worked decisively on the development of SARS-CoV-2 diagnostics. In July 2020, their SARS-CoV-2 assay for the GenomEra CDX system received the CE IVD approval for the European market. The results from the PCR-based COVID-19 test are available within 75 minutes. Because of the ease and rapidity of the system, many hospitals are using the assay for urgent samples such as testing the hospital personnel.

Thanks to our committed and competent team, we succeeded to develop and launch the new COVID-19 assay within a few months, and then increased our production capacity to meet the massive demand. Despite the global crisis and scarcity of some supply materials, we have managed to keep our production schedules and the reliability of delivery, says Mr. Erno Sundberg, the CEO of Abacus Diagnostica.

In addition to the COVID-19 assay, the company has been working on a four-in-one test kit for detecting influenza A, influenza B, RSV and SARS-CoV-2. The seasonal variation of both influenzas and RSV is evident (Figure 1). Every year these common viruses cause humane, social and economic burden for the people and the communities. In the Northern hemisphere, the flu season is approaching now on top of the COVID-19 pandemic. The health care system and clinical laboratories are already working on full capacity because of COVID-19, and that is a concern to Mr. Sundberg:

The COVID-19 testing is essential to manage and control the pandemic. When everyone is focused on COVID-19, there is a risk of delays on the diagnosis and treatment of other infections. It would benefit the patients and save the resources of the health care system to test more than one pathogen at a time. The flu season awaits in the shade of COVID-19 outbreak, and our new assay will help to bring it on sight as soon as it sets off.

Mr. Sundberg is confident with the company’s plans to seek the CE IVD approval for the multiplexed assay detecting influenzas, RSV and SARS-CoV-2. The time frame is set by the approaching flu season. Abacus Diagnostica is aiming for the market launch with well available, CE IVD approved four-in-one assay in November, in a good time before the flu season begins.
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Re: COVID-19 Tests | Wish List

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New multiplexed test with a low-cost sensor may enable at-home COVID diagnosis

10/1/20 ... nosis.aspx

One feature of the COVID-19 virus that makes it so difficult to contain is that it can be easily spread to others by a person who has yet to show any signs of infection. The carrier of the virus might feel perfectly well and go about their daily business--taking the virus with them to work, to the home of a family member, or to public gatherings.

A crucial part of the global effort to stem the spread of the pandemic, therefore, is the development of tests that can rapidly identify infections in people who are not yet symptomatic.

Now, Caltech researchers have developed a new type of multiplexed test (a test that combines multiple kinds of data) with a low-cost sensor that may enable the at-home diagnosis of a COVID infection through rapid analysis of small volumes of saliva or blood, without the involvement of a medical professional, in less than 10 minutes.

The research was conducted in the lab of Wei Gao, assistant professor in the Andrew and Peggy Cherng department of medical engineering. Previously, Gao and his team have developed wireless sensors that can monitor conditions such as gout, as well as stress levels, through the detection of extremely low levels of specific compounds in blood, saliva, or sweat.

Gao's sensors are made of graphene, a sheet-like form of carbon. A plastic sheet etched with a laser generates a 3D graphene structure with tiny pores. Those pores create a large amount of surface area on the sensor, which makes it sensitive enough to detect, with high accuracy, compounds that are only present in very small amounts. In this sensor, the graphene structures are coupled with antibodies, immune system molecules that are sensitive to specific proteins, like those on the surface of a COVID virus, for example.

Previous versions of the sensor were impregnated with antibodies for the hormone cortisol, which is associated with stress, and uric acid, which at high concentrations causes gout. The new version of the sensor, which Gao has named SARS-CoV-2 RapidPlex, contains antibodies and proteins that allow it to detect the presence of the virus itself; antibodies created by the body to fight the virus; and chemical markers of inflammation, which indicate the severity of the COVID-19 infection.

" This is the only telemedicine platform I've seen that can give information about the infection in three types of data with a single sensor. In as little as a few minutes, we can simultaneously check these levels, so we get a full picture about the infection, including early infection, immunity, and severity."

- Wei Gao, assistant professor in the Andrew and Peggy Cherng department of medical engineering

Established COVID-testing technologies usually take hours or even days to produce results. Those technologies also require expensive, complicated equipment, whereas Gao's system is simple and compact.

So far, the device has been tested only in the lab with a small number of blood and saliva samples obtained for medical research purposes from individuals who have tested positive or negative for COVID-19. Though preliminary results indicate that the sensor is highly accurate, a larger-scale test with real-world patients rather than laboratory samples must be performed, Gao cautions, to definitively determine its accuracy.

With the pilot study now completed, Gao next plans to test how long the sensors last with regular use, and to begin testing them with hospitalized COVID-19 patients. Following in-hospital testing, he would like to study the suitability of the tests for in-home use. Following testing, the device will need to receive regulatory approval before it is available for widespread use at home.

"Our ultimate aim really is home use," he says. "In the following year, we plan to mail them to high-risk individuals for at-home testing. And in the future, this platform could be modified for other types of infectious disease testing at home."
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Re: COVID-19 Tests | Wish List

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Rapid detection of SARS-CoV-2 with portable CRISPR-based mobile phone diagnostic test

10/4/20 ... -test.aspx

A large group of researchers from the United States reported developing an amplification-free CRISPR-based mobile phone assay for direct detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from nasal swabs. Their state-of-the-art paper is currently available on the medRxiv* preprint server.

An unrelenting coronavirus disease (COVID-19) pandemic caused by SARS-CoV-2 arose partly due to the challenges of identifying symptomatic, asymptomatic, and presymptomatic carriers of the virus – resulting in turn with their delayed isolation and massive global disease spread.

The current diagnostic gold standard is the quantitative reverse transcription-polymerase chain reaction (RT-qPCR), which is reliable and widely used for screening purposes. However, the situation in the United States shows a significant backlog, with 31% of nasal swab-based PCR tests necessitating more than four days to process.

This fact alone, coupled with a potential quick waning of the immunity after natural infection, highlights the need for rapid, point-of-care testing options that can also reliably detect SARS-CoV-2 genetic material.

In that regard, viral diagnostics may benefit from a successful bacterial strategy to destroy incoming bacteriophages (i.e., viruses that attack bacteria) and build an immunological memory, which is known as CRISPR. The latter can be exploited for the successful and quick detection of SARS-CoV-2.

To achieve the high sensitivity needed for testing purposes, current CRISPR diagnostic strategies primarily rely on pre-amplification of target ribonucleic acid (RNA) for subsequent detection by a Cas protein.

In a new and exciting study, a large research group reported the proof of concept of a rapid CRISPR-Cas13a-based diagnostic assay for the direct detection of SARS-CoV-2 RNA.

Optimizing a compact diagnostic device

In order to achieve this goal, the researchers first needed to optimize Cas13 activation through careful selection of CRISPR RNA complexes, as well as to develop a sensitive and transportable fluorescence detection system for this novel assay.

The simplicity of the approach was demonstrated by measuring fluorescence with a mobile phone camera in a close-packed device, comprised of low-cost laser lightning and collection optics.

The researchers have also combined complexes of CRISPR RNA and Cas13 (crRNAs) that target SARS-CoV-2 RNA to enhance the sensitivity and specificity of the proposed diagnostic solutions. They have also directly quantified viral load by utilizing enzyme kinetics.

Several key advances

A key advance of this approach is a successful demonstration of how these combinations of crRNAs can increase the sensitivity of Cas13a direct detection by including more Cas13a (which is a specific CRISPR-Cas13 subtype) per target RNA.

"By combining multiple crRNAs to increase Cas13a activation and analyzing the change in fluorescence over time rather than solely endpoint fluorescence, we are able to achieve SARS-CoV-2 viral RNA detection of ~100 copies/µL within 30 minutes", explained the study authors. "We also correctly identified all SARS-CoV-2 positive patient samples tested within 5 minutes", they add.

Another key advance is the possibility to translate the fluorescent signal into viral loads directly. Unlike previous diagnostic assays based on CRISPR technology, this one does not require pre-amplification of the viral genome. In other words, by directly detecting the viral RNA (without additional manipulations), we get quantitative RNA measurements instead of a simple, binary positive/negative result.

And of course, an additional important facet is that a simple mobile phone-based device can accurately read the direct-detection assay, avoiding the need for a bulky laboratory plate reader. This concept was already tried in previous molecular diagnostic approaches, primarily loop-mediated isothermal amplification.

Fast, portable and accurate

"Here we show that direct detection of SARS-CoV-2 RNA with CRISPR-Cas13a and a mobile phone offers a promising option for rapid, point-of-care testing", summarize study authors in this promising medRxiv paper.

Mobile phone cameras became highly sensitive tools, which (together with GPS, connectivity, and data-processing abilities) have turned them into attractive devices for point-of-care disease diagnosis, especially in low-resource regions.

Combined with mobile phone-based quantification, we can really see the potential in this approach to enable fast, portable, accurate, and low-cost options for point-of-care SARS-CoV-2 screening endeavors.

Going forward, direct detection by Cas13a (as outlined in detail in this paper) may be quickly modified to target the next emerging respiratory pathogen – hopefully in time to help curtail the global spread and another potentially devastating pandemic.
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Re: COVID-19 Tests | Wish List

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AbC-19 blood test detects COVID-19 antibodies 20 weeks after first symptoms/PCR positive result

10/5/20 ... esult.aspx

Serological antibody immunoassays are one of the essential tools to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In a new study, Immunoglobulin G (IgG) antibodies are detected in a large cohort, lasting up to 140 days. The researchers propose an alternative to reverse transcription-polymerase chain reaction (RT-PCR) positive status as a standard for assessing SARS-CoV-2 antibody assays and show robust performance metrics for the AbC-19 rapid test.

The COVID-19 (coronavirus disease 2019), caused by SARS-COV-2 the virus, has caused over 1 million deaths from over 35 million confirmed cases of infection globally over a short period (10 months). While the SARS-CoV-2 pandemic has necessitated rapid translation of knowledge from lab to clinic, an unprecedented interest has gained in serological immunoassays to detect antibodies to this novel virus. The dynamics of the immune response to SARS-CoV-2 is not yet clearly understood. It is essential to detect SARS-CoV-2 specific antibodies accurately. This is essential for building biobanks of convalescent sera for treatment, monitoring immune response to infection, and assessing responses to vaccination programs.

In their recent medRxiv* paper, Louise J Robertson et al. report the persistence of SARS-CoV-2 IgG up to 140 days (20 weeks) post-infection, across three antibody immunoassays and propose a standard for assessing SARS-CoV-2 antibody assays. The authors use a ‘pseudo gold standard’ cohort (n=348 positive, n=510 negative) to determine the sensitivity and specificity of the three commercial immunoassays (EuroImmun; Sens. 98.9% [97.7-99.7%]; Spec. 99.2% [98.4-99.8%]; Roche; Sens. 99.4% [98.6-100%]; Spec. (96.7% [95.1-98.2%]; Abbott; Sens. 86.8% [83.1-90.2%]; Spec. (99.2% [98.4-99.8%]). The UK-RTC AbC-19 lateral flow immunoassay shows a sensitivity of 97.70% (95.72%-99.34%) and specificity of 100% (100.00-100.00%). The study cohort included 880 people in Northern Ireland.

There is no clear gold standard for reference to assess SARS-CoV-2 immunoassays. RT-PCR is used as a reference standard; however, it is limited by a short temporal window for a positive diagnosis and exhibits potential for false-negative results. With lockdown measures and “flattening the curve” strategies, many cases were largely unconfirmed or undetected. The commercial serology immunoassays are laboratory-based and measure IgG antibody levels in plasma or serum. Alternatively, lateral flow immunoassays (LFIAs), requiring a finger-prick blood sample, can be used at home or POC (point-of-care).

Currently, in the UK, a limited number of laboratory-based chemiluminescence immunoassays are approved, including the Roche Elecsys AntiSARS-CoV-2 IgG/IgA/IgM against the SARS-CoV-2 Nucleocapsid antigenic region (Roche Diagnostics, Basel, Switzerland) and the Abbott SARS-CoV-2 IgG assay against the same antigenic region (Abbott Diagnostics, Abbott Park, IL, USA).

The authors use these tests to detect antibodies generated against the S1 domain of the spike antigenic protein of SARS-CoV-2, to characterize a large number (880) of pre-pandemic and pandemic COVID-19 blood samples from within Northern Ireland. IgG antibodies to both the spike protein and nucleocapsid protein are persistent even after 140 days after RT PCR positive status; the authors report a statistically significant decline over time, but the levels remain detectable at 140 days.

The authors call for further studies on large cohorts with sequential antibody immunoassays performed on symptomatic and non-symptomatic individuals as well as those with mild and severe COVID-19.

To assess sensitivity and specificity, they developed a ‘pseudo-gold-standard’ against which to analyze assays, which does not rely on a single test as reference. They report performance metrics for the UK-RTC AbC-19 rapid lateral flow immunoassay (LFIA) against a characterized panel of 304 positives established using the ‘pseudo gold standard’ system and 350 negative samples.

Their results show a strong correlation between all three immunoassays, with shortcomings in the Abbott system, suggesting an overestimated positive cut-off. Furthermore, they also present results of independent validation of the UK-RTC AbC-19 POC LFIA against a cohort of 304 known positives according to this ‘pseudo-gold-standard’ system and 350 known negative samples for IgG to SARS-CoV-2.

It is crucial to consider the prevalence when interpreting an assays’ sensitivity and specificity in a low prevalence scenario. Even slightly lowered performance metrics can result in large numbers of false-negative and false-positive results. The authors observe the high sensitivity of the AbC-19 LFIA - in which case, false-positives are unlikely, though false negatives may occur. This will underestimate true prevalence in a study.

For individuals, it may cause anxiety. However, a positive antibody test will help arrest infection spread.

In conclusion, the researchers report the longevity of antibodies against the SARS-CoV-2 virus in the plasma of a large cohort of individuals - lasting up to 140 days; and they have developed a ‘pseudo gold standard’ reference cohort against which to assess immunoassay performance.
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Feasibility of using dry reagents LAMP assay for SARS-CoV-2 detection

10/5/20 ... ction.aspx

Researchers evaluate a loop-mediated isothermal amplification (LAMP) assay using dry reagents for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). They found the test kit to be highly specific and sensitive. This method could enable point-of-care diagnosis in low-cost settings.

The rapid spread of the COVID-19 pandemic caused by the SARS-CoV-2 virus has necessitated the development of diagnostic methods that can quickly identify if a patient has been infected with the virus. Rapid detection of the virus is essential, as many of the symptoms of the infection, such as fever and cough, are similar to seasonal influenza or other respiratory illnesses. In addition, many patients have mild symptoms or are asymptomatic.

The most commonly used test is the real-time reverse transcription-polymerase chain reaction (RT-PCR). The method needs a special thermal cycler and precision optics to measure the emitted fluorescence from the samples. These requirements generally preclude its use in point-of-care (POC) settings.

Another method called the loop-mediated isothermal amplification (LAMP) method can amplify DNA under isothermal conditions with high specificity and sensitivity. This method is cheaper and faster than RT-PCR and has been used in POC testing of many diseases.

Testing LAMP assay with dry reagents

In a new study published on the preprint server medRxiv*, researchers evaluated the suitability of the Loopamp SARS-CoV-2 Detection kit from Eiken Chemica, Japan, for detecting SARS-CoV-2. This is a LAMP method that uses dry reagents for RNA amplification.

Dry reagents are more stable than liquid reagents, do not require a freezer for storage, and are easier to handle.

This will allow the method to be used at low-cost and developing countries, as the reagents can be stored in the refrigerator at about 4 °C and do not require stringent cold-chain transport and storage.

For evaluation of the LAMP method, the researchers first extracted RNA from nose and throat swabs from patients. They performed reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 detection kit from Eiken Chemical.

All the reagents, except the primers, are dried and immobilized inside the tube lid. The authors added the purified RNA, and the SARS-CoV-2 specific primer sets to the tube bottom and shook the tube several times to resuspend the enzyme and buffer. They incubated the mixture, collected at the bottom of the tube by a spin down, for 35 minutes at 62.5 °C.

To test the sensitivity of the kit, the authors tested 22 viruses, including SARS coronavirus, Middle East respiratory (MERS) syndrome coronavirus, other human coronaviruses, and influenza viruses.

They did not see any amplification of viruses other than SARS-CoV-2, indicating that the test was specific to the virus. The researchers confirmed these results using a turbidity assay and agarose gel electrophoresis analysis.

To define the sensitivity of the test, the researchers used in vitro transcribed RNA serially diluted in a buffer and used carrier RNA at a concentration of 50 ng/mL to define the detection limit.

The detection limit found by the authors was 10 copies/reaction using turbidity assays and color changes seen with the eye and using UV illumination. This is similar to or slightly higher than those for LAMP assays for SARS-CoV-2.

Clinical samples

The authors also evaluated their test using 24 clinical samples, which included three asymptomatic people who were in close contact with COVID-19 patients. The samples were collected between 7 March and 30 April 2020.

Using RT-PCR, 19 of the 24 samples resulted positive for SARS-CoV-2, whereas 15 samples showed positive results using the LAMP test evaluated by the authors.

The four false-negative samples, collected more than 7 days after the onset of the disease, had low copies of viral RNA of fewer than 10 copies/reaction, which is the detection limit of the new test. Two of the four false-negative samples were collected on days 18 and 20 to confirm the absence of the virus before discharge from the hospital.

Hence, the authors calculated the specificity, sensitivity, positive predictive value, and negative predictive value to be 100%, 78.9%, 100%, and 55.6%, respectively.

“Given that both turbidity assay and colorimetric changes detected all samples with more than 4.4 copies/reaction, we believe that this SARS-CoV-2 dry LAMP method is reliable for clinical use in diagnosing COVID-19,” write the authors.

A previous study has reported that SARS-CoV-2 RNA can be amplified in the LAMP assay without extraction. Incorporating such a method without RNA extraction could help a direct detection version of the test method for POC tests.
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Sebum changes in COVID-19 patients, for future testing and prognosis

10/5/20 ... nosis.aspx

The COVID-19 pandemic began in the last month of 2019 and spread rapidly throughout the world, causing over one million deaths so far, besides many tens of millions of confirmed infections. It is particularly dangerous because of its rapid and extensive infectivity combined with high mortality. Now, a new study published on the preprint server medRxiv* in September 2020 shows that ­­­­­­­­­­­­­­­­­­­sebum lipids change in COVID-19. This could help develop a cheap and straightforward method of non-invasive diagnosis.

Virus Testing Via PCR

The virus, SARS-CoV-2, enters the human body via the angiotensin-converting enzyme 2 (ACE2) receptor on the target host cells. Most symptoms relate to the respiratory system. A significant minority of cases will develop severe and hyperinflammatory features involving the lungs, leading to acute distress syndrome (ARDS), often associated with a cytokine storm.

Thus, the clinical manifestations of COVID-19 reflect both the direct harm caused by the virus infection and the immune response of the host. Mass testing is necessary to contain the virus and reduce the burden on the health services, usually involving the collection of upper respiratory samples and testing for viral RNA by polymerase chain reaction (PCR).

However, this approach has a sizable false-negative rate due to many factors and fails to offer prognostic value. Thus, research is on to assess the impact of the virus on the host rather than the virus itself, as an additional testing modality.

COVID-19 and the Sebum Lipidome

The current study focuses on the effect of COVID-19 on the sebum lipidome. Earlier studies have shown that patients with COVID-19 do have disrupted lipidomes, as shown by an examination of the blood plasma and nasopharyngeal swab lipids.

The researchers in the current study suggest that since dogs can be trained to sniff out COVID-19 patients, the skin of these individuals is likely to show a dysregulated lipidome. Thus, they say, “Lipidomics, therefore, offers a promising route to a better understanding of - and potentially diagnosis and prognosis for - COVID-19.”

The study centers on examining sebum, the greasy fluid secreted from the sebaceous glands in the skin, using liquid chromatography mass spectrometry. A sebum sample is painless and straightforward to obtain. The study of sebum lipids has been shown to yield characteristic features in illnesses like Parkinson’s Disease. Thus, the researchers sought to establish the sebum lipid patterns distinctive for COVID-19. This could be developed, if so, as a non-invasive sampling method for diagnostic testing in the future.

The COVID-19 International Mass Spectrometry (MS) Coalition was set up in May 2020 by a number of UK institutions to derive information on the virus at molecular level from infected individuals. This would help understand how the virus affects metabolic pathways so as to improve the diagnosis and treatment of COVID-19. The current study is part of the work of this coalition.

Study Group

The study included 67 participants, 30 with and 37 without clinical symptoms of COVID-19. All in the first group had a positive RT PCR test as well. The male-female ratio was slightly higher in the first group, perhaps because the infection is more severe in males.

The first group had a lower prevalence of chronic underlying illness, but diagnostic markers like CRP were higher. Lymphocyte counts were lower, however, while bilateral changes on the X-ray of the chest were more likely to occur in this group. Oxygen requirement was also higher, and they were more at risk of needing CPAP. They had higher odds of progressive disease and lower odds of survival.

Analysis by lipid class

The researchers found that COVID-19 patients had lower levels of triglycerides, diglycerides, and monoglycerides in the serum samples. This suggests a dysregulation of skin lipids. The differences between the levels of these lipids between the first and second groups were similar to those for CRP levels, and thus equally valuable as indicators of the presence of COVID-19.

Earlier work agrees on the existence of disrupted lipid levels in plasma, but the evidence is discordant as to whether lipid levels go up or down. Mild COVID-19, for instance, may be associated with elevated plasma triglyceride (TAG) levels, but this may decline with more severe disease. However, most of the lipid in the skin is synthesized locally rather than being supplied by the blood.

Population-level clustering analyses

Population-level clustering analyses (PCA) failed to show clustering of serum lipid concentrations by type, while OPLS-DA showed some insignificant and non-predictive separation of COVID-19 and non-COVID-19 individuals. When confounding factors like age, CRP, and lymphocyte count were adjusted for, they found that there was little improvement in the predictive or diagnostic value.

By itself, low lymphocyte counts were better strongly correlated with a diagnosis of COVID-19. Age was less important, indicating it does not affect the skin lipids significantly.

Classification by Comorbidity

The model performed better in four subsets when separate comorbidities and medication classified the sample.

These included those patients with a chronic illness that was treated by medication, namely, high cholesterol, type 2 diabetes mellitus (T2DM) and IHD, and those on statins. These subsets had better predictive power as well as the improved separation of COVID-19 positives from negatives. The reasons might include the increased homogeneity of these subsets concerning confounding factors.

Reasons for Increased Predictive Power

When patients are classified by treatment for comorbidity, it leads to a more uniform distribution in each subset. For instance, patients treated for high blood pressure or for T2DM are likely to be of the same range of ages, and even more so for those with high cholesterol. Sex-based subsets also provide adequate separation of cases from negatives.

The researchers postulate that if a more extensive set of patients and controls is used to allow better stratification, the predictive power will rise still more.

Another reason may be that medication use alters the effect of the confounding factors, reducing the patients to a more uniform baseline that is useful to estimate the disturbance resulting from the impact of COVID-19 on the lipidome. For instance, statins are classically used to treat high cholesterol and diabetes and ischemic heart disease as a preventive for better long-term outcomes. Analysis of the statin group yields good predictive power and separation.

Noting that this is a pilot study, the researchers say the risk of overfitting the model can be brought down by using more data for training the model and then validating the model on several sets of data.

Implications and Future Directions

The non-COVID-19 patients in this study were sampled in May, June, or July, and therefore had a lower incidence of other respiratory illnesses caused by seasonal respiratory viruses. This may have led to a potential absence of confounding factors since the latter could also cause lipid metabolism to register a change that could potentially prevent the identification of characteristic COVID-19 features.

In the UK, such viruses do circulate in fall and winter, and this must be considered in future research. Moreover, samples of sebum from COVID-19 patients taken over time will help to identify the time frame along which the sebum lipids become normal again following COVID-19 and the predictive power of these changes, which will determine its application in clinical or mass testing.

The study concludes, “COVID-19 infection leads to dyslipidemia in the stratum corneum.” Sebum lipidomics can help identify COVID-positive and negative patients with greater certainty if they are grouped by comorbidity. The ease with which sebum samples are obtainable, transported, and stored makes this a promising approach for sebum sampling for the diagnosis and prognosis of COVID-19.
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Wyss Institute’s technologies licensed to Agile Biodetection to aid COVID-19 diagnostic efforts

10/5/20 ... forts.aspx

The Wyss Institute for Biologically Inspired Engineering at Harvard University announced today that its nasopharyngeal swab and toehold switch technologies have been licensed to Alabama-based Agile Biodetection, which will use them to develop solutions for unmet diagnostic needs in the detection of the SARS-CoV-2 virus in environmental or clinical settings. The licensing agreement was coordinated by Harvard's Office of Technology Development (OTD) in accordance with the University's commitment to the COVID-19 Technology Access Framework.

The Wyss Institute's nasopharyngeal (nasal) swabs were developed in a multi-institutional and multi-disciplinary group effort led by Wyss Institute Senior Staff Engineer Richard Novak, Ph.D., and the Wyss' Founding Director Donald Ingber, M.D., Ph.D., as part of the Institute's collective response to the COVID-19 pandemic. Motivated by the serious shortage of swab devices for the collection of nasopharyngeal samples early in the pandemic, the researchers created a simple and effective device with advantages over other designs.

The Wyss swab device is fully injection-molded from a single material, and as such, can be mass manufactured in a one-step process that is faster, less expensive, and routinely used by a broad range of experienced medical device manufacturers worldwide. Conventional nasal swabs that are commonly used in infectious disease diagnostic medicine were designed 50 years ago, and are manufactured in two parts from different materials that then need to be assembled, sterilized and packaged in a multi-step process, which requires considerable time and expense.

In successful tests performed by academic collaborators and teaching hospitals, the unique nasal swab design was demonstrated to effectively collect SARS-CoV-2 genetic RNA material from the nostrils of patients and to be more comfortable than existing commercial products.

Toehold switches could come into play at the other end of the COVID-19 diagnostic process. Pioneered in the groups of Wyss Core Faculty members James Collins, Ph.D., and Peng Yin, Ph.D., they are synthetic nucleic acid-based devices that function as sensors for external stimuli ("inputs"), like RNA molecules derived from pathogenic viruses. When integrated into synthetic gene circuits, Toehold Switches can be designed to turn on a gene of interest, which can be a reporter signaling the presence of the environmental stimulus.

In their OFF state, these nanotechnological devices form a hairpin-like structure that specifically associates with and actively blocks the expression of a (reporter) target gene. Once an "input" RNA binds to their "toehold" region, the hairpin structure opens up and adopts an ON state to allow the protein-synthesizing machinery access to the target gene, which results in the synthesis of the actual signaling molecule.

In a series of proof-of-concept studies, the Collins and Yin teams have demonstrated toehold switches to function in living cells as computational devices that can assess and report complex combinations of environmental stimuli. They also have utilized them as key components of paper-based synthetic gene circuits that can be applied as diagnostics to sense and indicate different pathogens, including Ebola and Zika viruses. The versatile capabilities of Toehold Switches offer an opportunity for inexpensively and effectively surveilling the presence of pathogens with high sensitivity and specificity in different environmental settings, including working environments during the reopening phase of the pandemic.

Paving the way to real-world diagnostic solutions

Agile Biodetection is approaching the pandemic from an alternate viewpoint. Instead of testing patients for the virus, they're focusing on detecting SARS-CoV-2 in the environment, including on surfaces and touchpoints within high-traffic areas. They're applying a high-throughput cloud-based system to dissiminate the results of testing, making it faster and providing a leading indicator of the risk of infection.

" We believe that 3-pronged testing... of individuals, surfaces, and the environment, will be necessary to provide students, consumers and employees confidence to start the path back to normalcy."

-Kanti Sunkavalli, M.D., M.B.A., CEO of Agile Biodetection

Harvard co-led the creation of the COVID-19 Technology Access Framework, now endorsed by 23 other research institutions, to incentivize the rapid utilization of available technologies that can facilitate the diagnosis, treatment, and prevention of COVID-19 infection in the fight against the pandemic. Accordingly, the term-limited license agreement crafted by Harvard OTD grants Agile non-exclusive, royalty-free access to the Wyss nasal swab and Toehold Switch technologies for use in new SARS-CoV-2 detection approaches during the pandemic.

"This licensing agreement speaks to our Institute's ability to rapidly pivot and refocus our undivided attention on important and difficult problems in real-time as they arise, as we did with COVID-19. We are fully committed to help soften the blow of the pandemic in any way we can, and to help get bring normalcy into our lives again," said Ingber, who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children's Hospital, and Professor of Bioengineering at SEAS.
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NIST technique could improve detection of SARS-CoV-2 virus

10/5/20 ... virus.aspx

A multidisciplinary research team at the National Institute of Standards and Technology (NIST) has developed a way to increase the sensitivity of the primary test used to detect the SARS-CoV-2 virus, which causes COVID-19. Applying their findings to computerized test equipment could improve our ability to identify people who are infected but do not exhibit symptoms.

The team's results, published in the scientific journal Analytical and Bioanalytical Chemistry, describe a mathematical technique for perceiving comparatively faint signals in diagnostic test data that indicate the presence of the virus. These signals can escape detection when the number of viral particles found in a patient's nasal swab test sample is low. The team's method helps a modest signal stand out more clearly.

" Applying our technique could make the swab test up to 10 times more sensitive. It could potentially spot more people who are carrying the virus but whose viral count is too low for the current test to give a positive result."

-Paul Patrone, NIST physicist and co-author on the team's paper

The researchers' findings prove that the data from a positive test, when expressed in graphical form, takes on a recognizable shape that is always the same. Just as a fingerprint identifies a person, the shape is unique to this type of test. Only the shape's position, and importantly, its size, differ when graphed, varying with the quantity of viral particles that exist in the sample.

While it was known previously that the shape's position could vary, the team learned that its size can vary as well. Reprogramming test equipment to recognize this shape, regardless of size or location, is the key to improving test sensitivity.

The swab test employs a lab technique called quantitative polymerase chain reaction, or qPCR, to detect the genetic material carried by the SARS-CoV-2 virus. The qPCR technique takes any strands of viral RNA that exist in a patient's swab sample and then multiplies them into a far larger quantity of genetic material. Each time a new fragment of this material is made, the reaction releases a fluorescent marker that glows when exposed to light. It is this fluorescence that indicates the presence of the virus.

While the test method usually works well in practice, it can lack sensitivity to low viral particle counts. The test starts with the genetic material that is present and doubles it, then doubles it again, up to 40 times over, so that the fluorescent markers generate enough light to trigger a detector. Doubling, as anyone familiar with compound interest knows, is a powerful amplifier, growing slowly at first and then spiking to high numbers. The doublings produce a graph that is initially flat other than the bumps from systemic background noise, and eventually a telltale spike rises from it.

However, when the initial viral count is low, there may be false starts in the first few cycles. In these cases, even 40 doublings may not build a spike tall enough -- or a fluorescence bright enough -- to rise above the detection threshold. This issue can cause problems like inconclusive tests or "false negatives," meaning a person carries the virus but the test does not reveal it.

Preliminary studies indicate that the rate of false negatives may be as high as 30% in qPCR testing for COVID-19, including one study in which chest CT scans indicated positive cases where swab tests had not. Another study shows that asymptomatic and early-disease states are associated with up to 60 times fewer virus particles in patient samples. A JAMA study published in August supports the idea that asymptomatic carriers can spread the virus.

The NIST researchers found that the shape of a positive test graph -- a flat, noisy beginning followed by a spike -- is found even in data that currently does not trigger a positive test result. Their paper offers a formal proof that the shapes are mathematically "similar," akin to triangles that have the same angles and proportions despite being larger or smaller than one another. They apply this theoretical evidence in a routine that a computer can use to recognize the reference shape in the data.

"We're no longer constrained by having to pass a high detection threshold," Patrone said. "The spikes don't need to be large. They need to have the right shape."

Incorporating their findings into tests would immediately help the pandemic response, Patrone said, as it would help determine the number of asymptomatic and presymptomatic cases more accurately.

"In essence, lowering false negatives should help doctors and scientists get a better handle on the actual spread of the virus," he said. "There is a good chance that we're missing asymptomatic cases with the testing. The reduction we project in the number of viral RNA detected could pick up a significant number of asymptomatic cases."

The new test also would be unlikely to generate false positives because it would check that the curve was consistent with a reference shape, not merely that it crossed a detection threshold.

"In standard testing protocols, it is possible to get false positives -- for example, if background effects rise to the detection threshold and no one manually checks the result," Patrone said. "The likelihood of that happening in our analysis is very small because the math automatically rules out such signals."

Pandemic response workers would not need to do anything differently when collecting samples. Because the team's approach uses a mathematical algorithm applied after data collection, programmers could apply it by updating the lab equipment software with a few lines of computer code.

"Our work is a potentially easy fix because it's an advance in the data analysis," Patrone said. "It can easily be incorporated into the protocol of any lab or testing instrument, so it could have an immediate impact on the trajectory of the health crisis."
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Re: COVID-19 Tests | Wish List

Post by trader32176 »

Easier-to-use coronavirus saliva tests start to catch on

10/6/20 ... ch-on.aspx

As the coronavirus pandemic broke out across the country, health care providers and scientists relied on the standard method for detecting respiratory viruses: sticking a long swab deep into the nose to get a sample. The obstacles to implementing such testing on a mass scale quickly became clear.

Among them: Many people were wary of the unpleasant procedure, called a nasopharyngeal swab. It can be performed only by trained health workers, putting them at risk of infection and adding costs. And the swabs and chemicals needed to test for the virus almost immediately were in short supply.

Some places, like Los Angeles County, moved early to self-collected oral swabs of saliva and sputum, with the process supervised at drive-thru testing sites by trained personnel swathed in protective gear. Meanwhile, researchers began investigating other cheaper, simpler alternatives to the tried-and-true approach — including dribbling saliva into a test tube.

But the transition has not been immediate. Regulators and scientists are generally cautious about new, unproven technologies and have an understandable bias toward well-established protocols.

"Saliva is not a traditional diagnostic fluid," said Yale microbiologist Anne Wyllie, part of a team whose saliva-based test, called SalivaDirect, received emergency use authorization from the Food and Drug Administration in August. "When we were hit by a virus that came out of nowhere, we had to respond with the tools that were available."

Eight months into the pandemic, the move toward saliva screening is gaining traction, with tens of thousands of people across the country undergoing such testing daily. However, saliva tests still represented only a small percentage of the more than 900,000 tests conducted daily on average at the end of September.

Yale is providing its protocol on an open-source basis and recently designated laboratories in Minnesota, Florida and New York as capable of performing the test. Besides the Yale test, the FDA has authorized emergency use of several others, including versions developed at Rutgers University, the University of Illinois at Urbana-Champaign, the University of South Carolina and SUNY Upstate Medical University. A further advance, an at-home saliva test, could be headed for FDA authorization, too.

Since the start of the pandemic, the Trump administration's approach to testing has been hampered by missteps and controversy. As a key health agency during an unprecedented emergency, the FDA's effectiveness relies on public trust in how it balances the need for speed in authorizing innovative products, like saliva tests and vaccines, with ensuring safety and effectiveness, said Ann Keller, an associate professor of health policy at the University of California-Berkeley.

"You obviously want to get new tests into the mix quickly in order to address the emergency, but you still need to uphold your standards," Keller said. The White House's public pressure on the FDA has complicated the agency's efforts by undermining its credibility and independence, she said.

Respiratory viruses colonize areas inside the nasal cavity and at the back of the throat. Besides the nasopharyngeal approach, nasal samples obtained with shorter and less invasive swabs have proven effective for the coronavirus and have become widely adopted, although they also generally require a health care worker's involvement. The millions of rapid tests that will be distributed across the country, per a recent White House announcement, rely on nasal swabs.

In the early months of the pandemic, some studies reported significant levels of the virus in oral secretions. In a Hong Kong study published in February, for example, the virus was found in the saliva of 11 of 12 patients with confirmed coronavirus infection.

In Los Angeles, which began using the oral swab test in late March, more than 10,000 samples are collected per day, said Fred Turner, chief executive of Curative, the company that developed it.

Turner sees an advantage to the swabbing strategy. The self-swab procedure takes only 20 to 30 seconds, while producing enough saliva for testing can take people two to three minutes, and sometimes longer, he said. "That might not sound like much difference," Turner said, "but it is when you're trying to push 5,000 people through a test site."

Curative's three labs process tens of thousands of tests from jurisdictions across the country in addition to L.A., Turner said. A test developed at SUNY Upstate Medical University, which is expected to become available at state labs around New York, also uses an oral swab.

For the Curative test, a health care worker is supposed to oversee the sample collection —reminding people to cough to bring up fluids, for example. When investigators at the University of Illinois launched what they called a "Manhattan Project" to develop a saliva test by mid-June, they hoped to make it possible for people to visit a collection site, drool into a test tube, seal it and drop it off without the aid of a health care worker.

The university is now testing more than 10,000 people a day at its three campuses and is seeking to expand access to communities across the state and country, said chemistry professor Paul Hergenrother, who led the research team. Like the similar Yale test, it is being made freely available to other laboratories. The University of Notre Dame, in Indiana, recently adopted it.

Like tests using nasopharyngeal and other kinds of nasal swabs, these saliva tests are based on PCR technology, which amplifies small amounts of viral genetic material to facilitate detection. Both the Yale and University of Illinois tests have managed to simplify the process by eliminating a standard intermediate step: the extraction of viral RNA. Their protocols also don't require viral transport media, or VTM — the chemicals generally used to stabilize the samples after collection.

"You don't need swabs, you don't need health care workers, you don't need VTM, and you don't need RNA isolation kits," Hergenrother said.

In correspondence published in the New England Journal of Medicine, the Yale team reported detecting more viral RNA in saliva specimens than in nasopharyngeal ones, with a higher proportion of the saliva tests showing positive results for up to 10 days after initial diagnosis.

The National Basketball Association provided $500,000 in support for the Yale project, said David Weiss, the NBA's senior vice president for player matters. He said the Yale team's decision to eliminate the process of RNA extraction, which separates the genetic material from other substances that could complicate detection, involved trade-offs but did not compromise the value of the test.

"Any molecular test that has an RNA extraction step is almost by definition going to be more sensitive, but it will also be more expensive and take longer and use supplies that are in shorter supply," he said. "If we're trying to look at surveillance testing to open up schools and nursing homes, a test that's still very sensitive and a lot cheaper is an important innovation."

Prices for coronavirus tests vary widely, running upward of $100. Tests based on the Yale or University of Illinois protocols, which require only inexpensive materials, could be available for as little as $10. The Curative testing service, which includes collection and transportation of samples as well as the laboratory component, averages around $150 per test depending on volume, said Clayton Kazan, chief medical director of the L.A. County Fire Department, which uses the tests.

Despite the advances in sample collection, tests using PCR — polymerase chain reaction — technology still require laboratory processing. Researchers have been investigating other approaches, including saliva-based antigen tests, that could be self-administered at home and would provide immediate results. (While PCR can detect coronavirus genetic material, antigen tests look for viral proteins that can signify a current infection.)

At least one company has announced it is seeking emergency use authorization for a saliva antigen test, although two others have dropped plans to develop their own versions as infeasible, according to The New York Times. Meanwhile, scientists at Columbia University, the University of Wisconsin and elsewhere are investigating the use of saliva with other kinds of rapid-test technologies.

"There's tons of interest" in an at-home saliva test, noted Yvonne Maldonado, chief of pediatric infectious diseases at Stanford University School of Medicine.

"People really do want to get that pregnancy-type kit out there," she said. "You could basically send people a little packet with little strips, and you pull off a strip every day and put in under your tongue."
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Re: COVID-19 Tests | Wish List

Post by trader32176 »

Antigen testing detects SARS-CoV-2 better than PCR

10/6/20 ... n-PCR.aspx

A team of scientists from the United States demonstrates that rapid antigen tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more effective in determining the actual infection status in patients with coronavirus disease 2019 (COVID-19) than real-time polymerase chain reaction (RT PCR)-based tests. The study is currently available in the medRxiv* preprint server.

SARS-CoV-2, the causative pathogen of the COVID-19 pandemic, is a single-stranded RNA virus that rapidly spread from human to human primarily via respiratory droplets. The average incubation time for the virus is 5.8 days, which can be extended up to 14 days. Studies have shown that although viral RNA can be detected by polymerase chain reaction (PCR), no infectious virus (live virus) remains in patients’ specimens after around 8 days of symptom onset.

Regarding the detection of SARS-CoV-2, the US Food and Drug Administration (FDA) has recently approved antigen-based tests for the rapid diagnosis of COVID-19. These tests are a type of immunoassay that directly detects the presence of a target viral protein (antigen) in biological samples. There is evidence showing that compared to PCR-based test results, antigen-based test results correlate better with culture-based test results.

Current study objective

Given the significance of antigen-based testing in defining infectiousness, the current study aimed at evaluating the efficacy of antigen-based SARS-CoV-2 testing in differentiating contagious people from non-contagious people compared to that of PCR-based testing.

Current study design

The scientists performed both PCR-based and antigen-based tests using respiratory swab samples obtained from various regions across the United States. The results were compared with the SARS-CoV-2 TMPRSS2 culture, a sensitive virus culture test used as the reference method for determining infectiousness.

Important observations

Of 38 PCR-derived positive samples, 28 were positive, and 10 were negative in virus culture testing. By comparing antigen-based test results, the scientists observed that all samples except one were positive in both PCR-based and culture-based tests were also positive in the antigen-based test. Of 10 samples that were positive in PCR but negative in viral culture, two were positive in the antigen-based testing. These findings indicate that antigen-based test results correlate better with virus culture results.

In addition, the scientists found that of 38 PCR-positive samples, nine were negative in the antigen-based test. The viral RNA load was significantly lower in these 9 samples compared to that in other samples. By comparing viral RNA load with test type, the scientists observed no difference between the antigen test and viral culture in providing positive results; however, the PCR test showed minimal similarity with viral culture by generating positive results at much lower RNA loads.

Regarding test efficacy, the antigen test showed 96% sensitivity and 98% specificity, whereas the PCR test showed 100% sensitivity and 95% specificity. With a study prevalence of 11%, which was determined by virus culture-positive results (a total of 251 samples), the antigen test and PCR test showed positive predictive values of 90% and 73%, respectively.

Current study significance

The current study findings indicate that both antigen tests and PCR tests have almost equal sensitivity in detecting SARS-CoV-2 over a time period of less than 8 days post symptom onset. However, alike virus culture tests, antigen tests perform better in detecting the presence of the infectious virus in patients’ samples compared to PCR-based tests. It is possible to detect viral RNA in respiratory swab samples by PCR tests even after the complete attenuation of the infectious virus.

Studies have shown that in COVID-19 patients, the infectious virus cannot be detected in samples collected from the upper respiratory tract after 10 days of symptom onset. Therefore, the chance of viral transmission from these people is very less.

With a comparatively higher positive predictive value than PCR tests, antigen tests may be more effective in rapidly and timely differentiating contagious people from non-contagious people.
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