Wednesday, August 17, 2022

A Fast, Accurate, Equipment-free Diagnostic Test for SARS-CoV-2 and its Variants

More than two years into the pandemic, the virus that causes COVID—SARS-CoV-2—continues to spread worldwide. Testing for the virus and its variants can help limit transmission and inform treatment decisions, and is therefore an important pillar of the public health response.

Now, a team of researchers from Princeton University and the Broad Institute of MIT and Harvard have created an easy-to-use diagnostic test for COVID infection that is more sensitive than the commonly used at-home antigen tests, and that also allows for the rapid and specific detection of SARS-CoV-2 variants in point-of-care settings. The study was published May 30, 2022 in the journal Nature Biomedical Engineering.

“Viral infections are often challenging to detect – as we all realized in the early days of the SARS-CoV-2 pandemic,” said Cameron Myhrvold, an assistant professor in the Department of Molecular Biology at Princeton.

The improved test detects virus using a different mechanism than the more familiar clinic-based PCR or at-home antigen tests. Instead, the test detects virus using a technology known as CRISPR, which has found widespread use in gene editing. CRISPR originated from a system used by bacteria to detect and defend against viral infections, and is uniquely suited to rapidly identifying specific genetic sequences.

SHINEv.2 is a CRISPR-based diagnostic test for SARS-CoV-2 and its variants that can be performed with only five simple steps. Depicted is a version of SHINE v2 that reads out its results via a method similar to other viral home tests.

Myhrvold previously worked with Broad Institute member and Harvard University professor Pardis Sabeti to develop a CRISPR-based test that could be tailored to diagnose specific viral infections. In the wake of the pandemic’s first wave in 2020, Myhrvold’s team of researchers modified their test, which they called SHINE, to detect SARS-CoV-2 with remarkable sensitivity.

“The original version of SHINE was more of a lab-based test,” said Myhrvold. “We wanted to make it possible to perform outside of a lab, with the ultimate goal of enabling people to self-test or test at home.”

Jon Arizti-Sanz, a graduate student in medical engineering in the joint Harvard-MIT Health Sciences and Technology department and whom Myhrvold is mentoring, spearheaded a collaborative effort between the two scientists’ research teams to improve SHINE. First, the group focused on eliminating the need for specialized equipment to prepare patient samples for testing. Next, they optimized the test so that its reagents don’t need to be kept in a freezer, which ensures that the test can be easily transported long distances.

They dubbed the improved test “SHINEv.2.” After confirming that the altered test worked well in their own laboratory, the group sent a test kit to a laboratory in Nigeria to see whether it could survive a long period in transit and still retain its accuracy and sensitivity. It did. But then a new challenge arose for the team to address.

“While we were working on SHINEv.2, new SARS-CoV-2 variants emerged, so we wanted to make tests for them,” said Myhrvold.

The team was able to quickly adapt SHINEv.2 to discern between infections mounted by the Alpha, Beta, Delta or Omicron SARS-CoV-2 variants in patient samples, and say the test can be rapidly modified to pick up on any other variants that may arise.

“The data indicating this approach can be used to distinguish SARS-CoV-2 variants of concern by differential detection of genomic mutations could enable us to better prepare for the appearance of the next mutation,” said Tony Hu, director of the Center for Intelligent Molecular Diagnostics and Weatherhead Presidential Chair at Tulane University. Hu reviewed the manuscript for Nature Biomedical Engineering.

Because it tests for the different variants all at once, this version of the test needs one special, but relatively inexpensive, piece of equipment to read out its results. Nonetheless, SHINEv.2 is much faster to perform, and requires much less equipment and expertise, than current approaches used to identify SARS-CoV-2 variants. The team envisions that it would mainly be used at doctors’ offices, even ones with limited resources in remote locations, to help physicians determine which viral variant is afflicting their patient, and tailor therapies appropriately.

The team also wanted to create a version of their test that would be usable by people at home, which meant optimizing it so that it can be performed without any special equipment.

“We created a fully equipment-free version that can be performed by incubating the reaction using body heat—holding it in the underarm—or at room temperature,” said Myhrvold. “Our goal was to minimize the need for heating as much as possible.”

“The approach reported by authors could be a significant contribution for pandemic control by providing a method that requires minimal sample processing or heating,” Hu said.

The home test doesn’t discern between the different viral variants, but the minimal sample processing involved in this version of SHINEv.2 would be a boon to the home user—especially with a test that can accurately detect an infection with high sensitivity, picking up on cases that might be missed by other types of home tests. Myhrvold is optimistic that SHINEv.2 will soon be joining the front lines in the battle against SARS-CoV-2, and any other new pathogen that arises in the future.

Citation: Jon Arizti-Sanz, A’Doriann Bradley, Yibin B. Zhang, Chloe K. Boehm, Catherine A. Freije, Michelle E. Grunberg, Tinna-Solveig F. Kosoko-Thoroddsen, Nicole L. Welch, Priya P. Pillai, Sreekar Mantena, Gaeun Kim, Jessica N. Uwanibe, Oluwagboadurami G. John, Philomena E. Eromon, Gregory Kocher, Robin Gross, Justin S. Lee, Lisa E. Hensley, Bronwyn L. MacInnis, Jeremy Johnson, Michael Springer, Christian T. Happi, Pardis C. Sabeti  and Cameron Myhrvold. Simplified Cas13-based assays for the fast identification of SARS-CoV-2 and its variants. Nature Biomedical Engineering. 2022. DOI: 10.1038/s41551-022-00889-z

Funding: Funding for the work described in this story was provided by DARPA D18AC00006; the Flu Lab; donors through TED’s Audacious Project (the ELMA Foundation, MacKenzie Scott, the Skoll Foundation and Open Philanthropy); the ‘la Caixa’ Foundation (ID 100010434, code LCF/BQ/AA18/11680098); start-up funds from Princeton University; Agreement No. HSHQDC-15-C-00064 awarded to Battelle National Biodefense Institute (BNBI)by the Department of Homeland Security (DHS) Science and Technology (S&T) Directorate for the management and operation of the National Biodefense Analysis and Countermeasures Center (NBACC), a Federally Funded Research and Development Center; the Howard Hughes Medical Institute; Merck KGaA Future Insight Prize; and the National Institutes of Health (RO1 GM120122- 01, U01AI151812 and U54HG007480).

Grant Numbers: D18AC00006, 100010434, code LCF/BQ/AA18/11680098, HSHQDC-15-C-00064, RO1 GM120122- 01, U01AI151812, U54HG007480

Funders: DARPA, Flu Lab, ELMA Foundation, MacKenzie Scott, TED’s Audacious Project, Skoll Foundation, Open Philanthropy, ‘la Caixa’ Foundation, Princeton University, Battelle National Biodefense Institute, Department of Homeland Security Science and Technology Directorate for the management and operation of the National Biodefense Analysis and Countermeasures Center, National Institutes of Health, Howard Hughes Medical Institute, Merck KGaA Future Insight Prize

Source: Department of Molecular Biology, Princeton University (https://molbio.princeton.edu/) 

Wednesday, August 10, 2022

New Approaches to BCC detection Using Loop-Mediated Isothermal Amplification

The pharmaceutical, personal care and medical device industries have experienced significant BCC contamination events in recent years,. As such, I will be presenting an overview of BCC investigation and remediation case studies at this year's PDA Global Conference on Microbiology.  

Numerous detection methods are currently in use, including those described in USP <60> and rapid PCR and real-time quantitative PCR (qPCR) assays. However, there is a continued desire to develop more rapid and sensitive BCC detection strategies.

To meet this need, a team of scientists from FDA, academia and industry recently published a paper titled, "Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products."

Their abstract is reproduced below, and I encourage you to review the publication at your leisure.

Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products. Soumana Daddy Gaoh, Ohgew Kweon, Yong-Jin Lee, John J. LiPuma, David Hussong, Bernard Marasa and Youngbeom Ahn. Pathogens 2021, 10(9), 1071. 

Abstract

Simple and rapid detection of Burkholderia cepacia complex (BCC) bacteria, a common cause of pharmaceutical product recalls, is essential for consumer safety. In this study, we developed and evaluated a ribB-based colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of BCC in (i) nuclease-free water after 361 days, (ii) 10 μg/mL chlorhexidine gluconate (CHX) solutions, and (iii) 50 μg/mL benzalkonium chloride (BZK) solutions after 184 days. The RibB 5 primer specifically detected 20 strains of BCC but not 36 non-BCC strains. The limit of detection of the LAMP assay was 1 pg/μL for Burkholderia cenocepacia strain J2315. Comparison of LAMP with a qPCR assay using 1440 test sets showed higher sensitivity: 60.6% in nuclease-free water and 42.4% in CHX solution with LAMP vs. 51.3% and 31.1%, respectively, with qPCR. These results demonstrate the potential of the ribB-based LAMP assay for the rapid and sensitive detection of BCC in pharmaceutical manufacturing.

Monday, August 8, 2022

EU Methods for the Detection and Characterisation of SARS-CoV-2 Variants Document Updated

The European Centre for Disease Prevention and Control has recently published its second edition of the "Methods for the detection and characterisation of SARS-CoV-2 variants" document

In the past year, several SARS-CoV-2 variants of concern (VOCs) have emerged and it is of key importance o monitor their circulation in all countries. Whole Genome Sequencing (WGS), or at least complete or partial spike (S)-gene sequencing, is the best method for characterising a specific variant. Alternative methods, such as diagnostic screening nucleic acid amplification technique (NAAT)-based assays, have been developed for early detection and pre-screening to allow prevalence calculation of VOCs, variants of interest (VOI) and variants under monitoring (VUM). Many of these methods can accurately identify the different variants, while others will require confirmation by sequencing of at least the complete or partial S-gene genomic region in a subset of samples.

Genomic monitoring should be integrated into the overall respiratory virus surveillance strategies. Specific objectives for the detection and identification of variants include assessment of the circulation of different SARS-CoV-2 variants in the community by selecting representative samples for sequencing, and genetic characterisation to monitor virus evolution and inform vaccine composition decisions or outbreak analyses. When NAAT-based assays are used, confirmatory sequencing of at least a subset of samples should be performed to use these assay results as indicators of community circulation of virus variants, particularly VOCs. Before introducing a new testing method or a new assay, a validation and verification exercise should be carried out to ensure that the laboratory testing system is reliably detecting the circulating viruses. Variant typing results should be reported to The European Surveillance System (TESSy) and SARS-CoV-2 consensus sequences should be deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database, or other public databases. If available, related sequencing raw data should be deposited in the European Nucleotide Archive (ENA) and raw data, if available, in the European Nucleotide Archive (ENA). This should be done in a timely manner (ideally on a weekly basis).

This document was developed by technical experts from ECDC and the World Health Organization (WHO) European Region and previous versions have been reviewed by experts at WHO’s referral laboratories and in the SARS-CoV-2 Characterisation Working Group.

New in this Update

- Information related to detection assays specific for Omicron variants BA.4 and BA.5 has been included.
- The chapter on rapid antigen detection tests (RADT) has been updated and includes available information on their performance for Omicron variants.
- The chapter on neutralisation assays has been updated with information on the isolation of SARS-CoV-2 BA.4/5 variants.

The document may be downloaded here: 

https://www.ecdc.europa.eu/sites/default/files/documents/Methods-for-the-detection-char-SARS-CoV-2-variants_2nd%20update_final.pdf

Wednesday, August 3, 2022

Identifying Bacteria Using Optical Properties of Nanometer-Scaled Metal-Organic Hybrids

A recent study published in Analytical Chemistry proposes a strategy for optical detection of multiple bacterial species based on the optical properties of nanohybrid structures of polymer-coated metal nanoparticles. A review of this exciting research was discussed in AZO Optics

Rapid detection of bacteria is essential due to the rise in antibiotic-resistant microbes, the global food trade, and their application in pharmaceutical, bioremediation, and food production. Optical detection techniques have piqued the curiosity of researchers due to their potential for fast, high-throughput, non-destructive, amplification-free identification.

Developments in Bacterial Detection Techniques

Several bacterial species are useful for enhancing safety and quality of life in medication, food and energy production; yet, some bacteria are dangerous.

Bacterial identification tests performed in the food, environmental and medical field must satisfy selectivity, sensitivity, cost, and speed standards. Recent years have seen extensive research into the development of bacterial testing, ranging from absorption, luminescence, or current response-based detections to the integration of spectroscopy or microscopy and deep learning.

While these advancements have numerous advantages, they require adequate development time to replace conventional approaches.

Challenges of Conventional Bacterial Detection Techniques

Although conventional bacterial tests offer advantages, they also present various obstacles.

Culturing

Culturing is a popular method for identifying bacterial species based on their biological activity. However, results take at least one day because of the culture time.

Gram staining

Gram staining can be done more quickly than culture. It distinguishes between gram-negative and gram-positive bacteria under a microscope but cannot distinguish bacterial species.

Fluorescent labeling

Fluorescent labeling detects dye-conjugated antibody-labeled bacteria using flow cytometry or a microscope. It has issues related to intensity adjustment and limited fluorescence lifespan.

Lateral flow test

The lateral flow test uses antibody-conjugated gold nanoparticles (AuNP) to label target bacteria for naked-eye detection. The label does not fade, based on the AuNP's localized surface plasmon resonance. Stable inspection is easier with the lateral flow test than fluorescent labeling. However, due to the label's low optical intensity, the considerable antigen must be cultured to see the color.

Using Nanometer-Scaled Metal−Organic Hybrids to Detect Bacteria

Researchers used the optical properties of nanometer-scaled metal-organic hybrids to identify various bacteria in their study.

Metal nanoparticles (NPs) are valuable for optical detection and have strong affinities to biological components. Darkfield microscopy (DFM) was utilized to investigate scattering light induced by target substances due to its ability to observe metal nanoparticles smaller than the theoretical resolution limit.

A reaction system that autonomously controls nanostructure production was developed using aniline and metal ions to produce organic metal NHs.

E. coli O157, E. coli O26, and S. aureus were added to the mixture to generate an assessment solution with 13% bacteria density of the total cells. The capacity of NHs to mark individual cells was investigated using sample suspensions isolated from rotting chicken mince.

A dark field microscope and a field emission scanning electron microscope helped examine the mixture of the antibody-conjugated NH dispersion and bacterial solution. Images from a dark field microscope were captured using an optical microscope equipped with a halogen lamp, darkfield condenser, and a charge-coupled device camera.

The light-scattering spectra were recorded with a small grating spectrometer connected via an optical fiber to the dark field microscope. Focusing on the NH labels' light-scattering features helped identify the bacterial species.

Important Findings of the Study

Organic metal NHs are an excellent identification tool, facilitating quantitative and qualitative investigations of bacterial species in the same reaction area.

The optical properties of the nanohybrid structures (NHs) depend heavily on the individual metal elements of nanoparticles.

The rate of false negatives was estimated to be around 6%, while false positives were not confirmed.

Integrating antibodies into NHs leads to the binding of antigens to the cells, allowing bacteria to be identified by light scattering. Multiple bacterial species deposited on a slide were recognized within one field of view of a dark field microscope using scattered light colors.

Future Developments

There are currently no rapid techniques for detecting several bacterial species in a small number of samples. However, the proposed approach will allow for the simultaneous identification of many bacterial species in a single reaction area, which is not currently attainable with current technology.

The advancement of bacterial testing methods will improve quality and safety in various sectors of our life, including food production, medicine, and energy harvesting.

Reference

Tanabe, S., Itagaki, S., Matsui, K., Nishii, S., Yamamoto, Y., Sadanaga, Y., & Shiigi, H. (2022). Simultaneous Optical Detection of Multiple Bacterial Species Using Nanometer-Scaled Metal–Organic Hybrids. Analytical Chemistry. https://pubs.acs.org/doi/10.1021/acs.analchem.2c01188

Source: AZO Optics.

Friday, July 22, 2022

Scientists Develop Optical Microring Resonator for the Rapid Detection of Ebola

A new tool can rapidly and reliably detect the presence of Ebola virus in blood samples at lower concentrations than existing tests, researchers from the US report. The device has the potential to help control future outbreaks of the deadly infection.

Ebola virus disease is a viral haemorrhagic fever that is estimated to kill up to 89% of those who contract it. It is spread through contact with the blood, bodily fluids or organs of an infected person or animal. First discovered following two simultaneous outbreaks in Nzara, in South Sudan, and Yambuku, in the Democratic Republic of the Congo, it has since led to dozens of outbreaks in the tropical regions of sub-Saharan Africa. The worst outbreak to date occurred in West Africa between late 2013 and early 2016, and is estimated to have caused 11,323 deaths.

In recent years, a selection of vaccines and effective therapies for Ebola have been developed. Unfortunately, however, they are not widely available. Accordingly, health officials typically combat the disease by attempting to contain outbreaks, an approach that relies on being able to quickly identify infections and inhibit further transmission. This is a challenge though, as Ebola symptoms – body aches, bleeding, diarrhoea and fever – are highly nonspecific, meaning that it can be easily mistaken for other viral infections or malaria.

Existing tests for the disease, meanwhile – which include PCR-based techniques, lateral flow assays and enzyme-linked immunosorbent assays (ELISAs) – are limited by lengthy assay times, and the need for additional electronics for sample processing, trained technicians and even cold chain custody. In addition, they tend not to be very sensitive until the virus has had days to multiply to high levels in the body.

In this latest study, clinical pathologist Abraham Qavi of Washington University in St Louis and his colleagues propose an alternative based on optical microring resonators, a type of whispering gallery mode device that can be used for highly sensitive molecular detection.

Such tools take their name from the effect originally discovered for sound waves in the Whispering Gallery in London’s St Paul’s Cathedral. Words whispered against the wall of the dome can be heard clearly more than 30 m away, thanks to the way in which sound waves travel around the concave surface. This is an example of the principle of acoustic resonance – a phenomenon that can also be seen with light waves at a much smaller scale.

Explaining how their whispering gallery mode device can detect the presence of tiny amounts of Ebola-related molecules in blood samples, Qavi says: “We trap light in the resonators and use resonance to enhance and boost our signal. By monitoring where this resonance wavelength occurs, we can tell how much of the molecule we have.”

The molecule in question is a sensitive antibody developed to react to a soluble glycoprotein released by the Ebola virus. This protein is also key to current diagnostic tests for Ebola — but the new antibody is capable of detecting it at lower levels. In tests on blood from infected animals, the microring resonator devices could detect the diagnostic glycoprotein as early as, or earlier than, the current leading tests. The test, which only took 40 min, also provided information on the viral glycoprotein concentration. This information could potentially be used to tailor treatment plans for individual patients.

“Any time you can diagnose an infection earlier, you can allocate healthcare resources more efficiently and promote better outcomes for the individual and the community,” Qavi says. “Using a biomarker of Ebola infection, we’ve shown that we can detect Ebola in the crucial early days after infection. A few days makes a big difference in terms of getting people the medical care they need and breaking the cycle of transmission.”

“Rapid, biosensor-based assays are needed to deal with a myriad of global health concerns, among them the detection of virus infections with the potential to spread across the globe,” says Frank Vollmer, a physicist from the University of Exeter, UK, who was not involved in the new study. Whispering gallery mode sensors, he explained, have emerged as one of the most sensitive and multiplexed biosensor technologies that can address this need.

He added: “[The researchers] impressively combine the high sensitivity and multiplexed readout of the whispering gallery mode sensor with the specific detection of the Ebola virus glycoprotein in patient samples – providing the real-world whispering gallery mode biosensor application that can save lives.”

With their initial study complete, the researchers are now looking to miniaturize the device and test their diagnostic approach on infected individuals.

The study is described in Cell Reports Methods.

Source: Physics World 

Thursday, July 7, 2022

Researchers Pioneer A New Way To Detect Microbial Contamination In Cell Cultures

Researchers from the Critical Analytics For Manufacturing Personalized-Medicine (CAMP) interdisciplinary research group at the Singapore-MIT Alliance for Research and Technology (SMART), MIT’s research enterprise in Singapore, have developed a new method of detecting adventitious microbial contamination in mesenchymal stromal cell (MSC) cultures, ensuring the rapid and accurate testing of cell therapy products intended for use in patients. Utilizing machine learning to predict if a culture is clean or contaminated in near-real time, this breakthrough method can be used during the cell manufacturing process, compared to less efficient end-point testing.

Cell therapy has, in recent years, become a vital treatment option for a variety of diseases, injuries, and illnesses. By transferring healthy human cells into a patient’s body to heal or replace damaged cells, cell therapy has shown increasing promise in effectively treating cancers, autoimmune diseases, spinal cord injuries, and neurological conditions, among others. As cell therapies advance and hold the potential to save more lives, researchers continue to refine cell culture manufacturing methods and processes to ensure the safety, efficiency, and sterility of these products for patient use.

The anomaly-detection model developed by CAMP is a rapid, label-free process analytical technology for detecting microbial contamination in cell cultures. The team’s research is explained in an oral abstract “Process Development and Manufacturing: Anomaly Detection for Microbial Contamination In Mesenchymal Stromal Cell Culture,” published recently in the journal Cytotherapy.

The machine learning model was developed by first collecting sterile cell culture media samples from a range of MSC cultures of different culture conditions. Some of the collected samples were spiked with different bacteria strains at different colony-forming units, a measurement of the estimated concentration of microorganisms in a test sample. The absorbance spectra of the sterile, unspiked and bacteria-spiked samples were obtained with ultraviolet-visible spectrometry, and the spectra of the sterile samples were used to train the machine learning model. Testing the model with a mixture of sterile and bacteria-spiked samples demonstrated the model’s performance in accurately predicting sterility.

“The practical application of this discovery is vast. When combined with at-line technologies, the model can be used to continuously monitor cultures grown in bioreactors at Good Manufacturing Practice (GMP) facilities in-process,” says Shruthi Pandi Chelvam, lead author and research engineer at SMART CAMP who worked with Derrick Yong and Stacy Springs, SMART CAMP principal investigators, on the development of this method. “Consequently, GMP facilities can conduct sterility tests for bacteria in spent culture media more quickly with less manpower under closed-loop operations. Lastly, patients receiving cell therapy as part of their treatment can be assured that products have been thoroughly evaluated for safety and sterility.”

During the process of cell therapy manufacturing, this anomaly-detection model can be used to detect the presence of adventitious microbial contamination in cell cultures within a few minutes. This in-process method can help to save time and resources, as contaminated cultures can immediately be discarded and reconstructed. This method provides a rapid alternative to conventional sterility tests and other microbiological bacteria detection methods, often taking a few days and almost always performed on finished products.

“Our increased adoption of machine learning in microbial anomaly detection has enabled us to develop a unique test which quickly performs in-process contamination monitoring, marking a huge step forward in further streamlining the cell therapy manufacturing process. Besides ensuring the safety and sterility of cell products prior to infusion in patients, this method also offers cost and resource effectiveness for manufacturers, as it allows for decisive batch restarting and stoppage should the culture be contaminated,” adds Yie Hou Lee, scientific director of SMART CAMP.

Moving forward, CAMP aims to develop an in-process monitoring pipeline in which this anomaly detection model can be integrated with some of the in-house at-line technologies that are being developed, which would allow for periodic culture analysis using a bioreactor. This would open the possibilities for further, long-term experimental studies in continuous culture monitoring.

Lead author Shruthi Pandi Chelvam also won the Early Stage Professionals Abstract Award, which is presented to three outstanding scholars, and abstracts are scored through a blinded peer-review process. The research was also accepted for the oral presentation at the 2022 International Society for Cell and Gene Therapy (ISCT) conference, a prestigious event in cell and gene therapies.

“This team-based, interdisciplinary approach to technology development that addresses critical bottlenecks in cell therapy manufacturing — including rapid safety assessment that allows on intermittent or at-line monitoring of plausible adventitious agent contamination — is a hallmark of SMART CAMP’s research goals,” adds MIT’s Krystyn Van Vliet, who is associate vice president for research, associate provost, a professor of materials science and engineering, and co-lead of SMART CAMP with Hanry Yu, professor at the National University of Singapore.

The research is carried out by SMART and supported by the National Research Foundation (NRF) Singapore under its Campus for Research Excellence And Technological Enterprise (CREATE) program. The study collaborated with a team from the Integrated Manufacturing Program for Autologous Cell Therapy, one of the sister programs in the Singapore Cell Therapy Advanced Manufacturing Program, of which CAMP is a part, to help develop an automated sampling system. This technology would integrate into the anomaly detection model.

CAMP is a SMART interdisciplinary research group launched in June 2019. It focuses on better ways to produce living cells as medicine, or cellular therapies, to provide more patients access to promising and approved therapies. The investigators at CAMP address two key bottlenecks facing the production of a range of potential cell therapies: critical quality attributes (CQA) and process analytic technologies (PAT). Leveraging deep collaborations within Singapore and MIT in the United States, CAMP invents and demonstrates CQA/PAT capabilities from stem to immune cells. Its work addresses ailments ranging from cancer to tissue degeneration, targeting adherent and suspended cells, with and without genetic engineering.

CAMP is the R&D core of a comprehensive national effort on cell therapy manufacturing in Singapore.

SMART was established by MIT in partnership with the NRF in 2007. SMART is the first entity in CREATE developed by NRF. SMART serves as an intellectual and innovation hub for cutting-edge research interactions of interest to both MIT and Singapore. SMART currently comprises an Innovation Center and five IRGs: Antimicrobial Resistance (AMR), CAMP, Disruptive and Sustainable Technologies for Agricultural Precision (DiSTAP), Future Urban Mobility (FM), and Low Energy Electronic Systems (LEES).

UTSW Researchers Develop Rapid COVID-19 Test to Identify Variants in Hours

In just a few hours, UT Southwestern scientists can tell which variant has infected a COVID-19 patient – a critical task that can potentially influence treatment decisions but takes days or weeks at most medical centers.

Last year, pathologist Jeffrey SoRelle, M.D., and colleagues developed CoVarScan, a rapid COVID-19 test that detects the signatures of eight hotspots on the SARS-CoV-2 virus. Now, after testing CoVarScan on more than 4,000 patient samples collected at UT Southwestern, the team reports in Clinical Chemistry that their test is as accurate as other methods used to diagnose COVID-19 and can successfully differentiate between all current variants of SARS-CoV-2. 

“Using this test, we can determine very quickly what variants are in the community and if a new variant is emerging,” said Dr. SoRelle, Assistant Professor of Pathology and senior author of the study. “It also has implications for individual patients when we’re dealing with variants that respond differently to treatments.”

The testing results at UT Southwestern’s Once Upon a Time Human Genomics Center have helped public health leaders track the spread of COVID-19 in North Texas and make policy decisions based on the prevalence of variants.  Doctors have also used the results to choose monoclonal antibodies that are more effective against certain strains infecting critically ill COVID-19 patients.

While a number of other tests for COVID-19 exist, they generally detect either a fragment of SARS-CoV-2 genetic material or small molecules found on the surface of the virus, and don’t provide information to identify the variant. In addition, many researchers worry that these tests aren’t accurate in detecting some variants – or may miss future strains. To determine which variant of COVID-19 a patient has, scientists typically must use whole genome sequencing, which is time-consuming and expensive, relying on sophisticated equipment and analysis to spell out the entire RNA sequence contained in the viruses.

In early 2021, Dr. SoRelle and his colleagues at UT Southwestern wanted to track how well current tests were detecting emerging variants of SARS-CoV-2. But they realized that sequencing a lot of specimens would not be timely or cost-effective, so they designed their own test, working in the McDermott Center Next Generation Sequencing Core, part of the Eugene McDermott Center for Human Growth and Development directed by Helen Hobbs, M.D., Professor of Internal Medicine and Molecular Genetics.

CoVarScan hones in on eight regions of SARS-CoV-2 that commonly differ between viral variants. It detects small mutations – where the sequence of RNA building blocks varies – and measures the length of repetitive genetic regions that tend to grow and shrink as the virus evolves. The method relies on polymerase chain reaction (PCR) – a technique common in most pathology labs – to copy and measure the RNA at these eight sites of interest. 

To test how well CoVarScan works, Dr. SoRelle’s team ran the test on more than 4,000 COVID-19-positive nasal swab samples collected at UT Southwestern from April 2021 to February 2022 – from patients both with and without symptoms. The tests were validated with the gold-standard whole genome sequencing, and the results were used by doctors to choose treatments in some critically ill COVID-19 patients.  

Compared to whole genome sequencing, CoVarScan had 96% sensitivity and 99% specificity. It identified and differentiated Delta, Mu, Lambda, and Omicron variants of COVID-19, including the BA.2 version of Omicron, once known as “stealth Omicron” because it did not show up on some tests designed to detect only the Omicron strain.

“A common critique of this kind of test is that it requires constant adjustment for new variants, but CoVarScan has not needed any adjustment in more than a year; it is still performing very well,” said Dr. SoRelle. “In the future, if we did need to adjust it, we could easily add as many as 20 or 30 additional hotspots to the test.”

Dr. SoRelle plans to continue developing CoVarScan as a commercial test and has a pending patent application based on this work. As the inventor of the genotyping PCR test for variants, Dr. SoRelle is entitled to income from its use.

Other UTSW researchers who contributed to this study include Andrew Clark, Zhaohui Wang, Emily Ostman, Hui Zheng, Huiyu Yao, Brandi Cantarel, Mohammed Kanchwala, Chao Xing, Li Chen, Pei Irwin, Yan Xu, Dwight Oliver, Francesca Lee, Jeffrey Gagan, Laura Filkins, Alagarraju Muthukumar, Jason Park, and Ravi Sarode.

Dr. Hobbs holds the 1995 Dallas Heart Ball Chair in Cardiology Research, the Philip O’Bryan Montgomery, Jr., M.D. Distinguished Chair in Developmental Biology, and the Eugene McDermott Distinguished Chair for the Study of Human Growth and Development. Dr. Sarode holds the John H. Childers, M.D. Professorship in Pathology.

About UT Southwestern Medical Center

UT Southwestern, one of the nation’s premier academic medical centers, integrates pioneering biomedical research with exceptional clinical care and education. The institution’s faculty has received six Nobel Prizes, and includes 26 members of the National Academy of Sciences, 17 members of the National Academy of Medicine, and 14 Howard Hughes Medical Institute Investigators. The full-time faculty of more than 2,900 is responsible for groundbreaking medical advances and is committed to translating science-driven research quickly to new clinical treatments. UT Southwestern physicians provide care in more than 80 specialties to more than 100,000 hospitalized patients, more than 360,000 emergency room cases, and oversee nearly 4 million outpatient visits a year.

Tuesday, June 21, 2022

Rapid Ebola Diagnosis May Be Possible With New Technology

A new tool can quickly and reliably identify the presence of Ebola virus in blood samples, according to a study by researchers at Washington University School of Medicine in St. Louis and colleagues at other institutions.

The technology, which uses so-called optical microring resonators, potentially could be developed into a rapid diagnostic test for the deadly Ebola virus disease, which kills up to 89% of infected people. Since it was discovered in 1976, Ebola virus has caused dozens of outbreaks, mostly in central and west Africa. Most notable was an outbreak that began in 2014 and killed more than 11,000 people in Guinea, Sierra Leone and Liberia; in the U.S., the virus caused 11 cases and two deaths. A rapid, early diagnostic could help public health workers track the virus’ spread and implement strategies to limit outbreaks.

The study — which also involved researchers from the University of Michigan, Ann Arbor, and Integrated Biotherapeutics, a biotech company — is published June 8 in Cell Reports Methods.

“Any time you can diagnose an infection earlier, you can allocate health-care resources more efficiently and promote better outcomes for the individual and the community,” said co-first author Abraham Qavi, MD, PhD, a postdoctoral researcher at Washington University. “Using a biomarker of Ebola infection, we’ve shown that we can detect Ebola infection in the crucial early days after infection. A few days makes a big difference in terms of getting people the medical care they need and breaking the cycle of transmission.”

Ebola virus is transmitted by contact with bodily fluids. It causes fever, body aches, diarrhea and bleeding — nonspecific symptoms that easily can be mistaken for other viral infections or for malaria. In recent years, vaccines and effective therapies for Ebola have been developed, but they are not widely available. Instead, health officials control the deadly virus by containing outbreaks. The strategy relies on quickly identifying infected people and preventing transmission by encouraging caregivers to wear protective gear.

Qavi had previously worked with Ryan C. Bailey, PhD, the Robert A. Gregg Professor of Chemistry at the University of Michigan and a co-senior author on this paper, to co-develop optical microring resonators, a kind of whispering gallery mode device used for molecular detection. The name comes from the Whispering Gallery at St. Paul’s Cathedral in London. A whisper uttered on a walkway in the dome above the nave can be heard clearly more than 100 feet away because the sound waves increase in amplitude as they bounce around the circular wall. The 18th century builders accidentally constructed a giant demonstration of the principle of acoustic resonance, in which sound waves increase in amplitude if they interact in precisely the right way. The same phenomenon occurs with light waves on a much smaller scale.

When Qavi joined the lab of co-senior author Gaya K. Amarasinghe, PhD — an Ebola expert and the Alumni Endowed Professor of Pathology & Immunology and a professor of biochemistry & molecular biophysics and of molecular microbiology at Washington University — they decided to apply the technology to create a better diagnostic test for Ebola. Qavi teamed up with Bailey, co-first author Krista Meserve, a graduate student in Bailey’s lab, and co-author Lan Yang, PhD, the Edwin H. and Florence G. Skinner Professor of Electrical & Systems Engineering at Washington University’s McKelvey School of Engineering, to develop a tool that could detect tiny amounts of Ebola-related molecules in blood samples using microring resonators.

“We trap light in the resonators and use resonance to enhance and boost our signal,” Qavi said. “By monitoring where this resonance wavelength occurs, we can tell how much of the molecule we have.”

The key was finding the right molecule. Current diagnostic tests detect the virus’s genetic material or a glycoprotein — a protein covered in sugar — produced by the virus. But they aren’t reliable until the virus has multiplied to high levels in the body, a process that can take days. Co-senior author Frederick Holtsberg, PhD, vice president of manufacturing and bioanalytics at Integrated Biotherapeutics, developed a highly sensitive antibody capable of detecting the viral soluble glycoprotein at low levels.

The researchers incorporated the antibody into their device and tested it using blood from infected animals. They found their technique could detect the glycoprotein as early as or earlier than the most sensitive test for viral genetic material. Importantly, the technology also allowed them to quantify the amount of viral glycoprotein in the blood. The higher the level, the worse the infected animals fared. Moreover, the test only took 40 minutes start to finish.

“Looking at these data, we can say, ‘If you’re above these levels, your chance of survival is low; if you’re below it, your chance of survival is high’,” Qavi said. “We still have to validate this in infected individuals, but if it holds up, doctors could use this information to tailor treatment plans for individual patients and allocate scarce medications to the patients most likely to benefit.

“We’ve shown the fundamental science works,” he added. “Now it’s just an issue of miniaturizing the devices and taking them into the field.”

Monday, June 20, 2022

Raman Spectroscopy as an Alternative to the Conventional Sterility Test

A study combining Raman spectroscopy with PLS-DA multivariate analysis achieved fast and non-invasive detection of contaminated drug products within vials.

Researchers have demonstrated the potential of a fast and non-invasive approach to detect pharmaceutical products contaminated with low levels of bacteria within their vials. The technique uses dispersive Raman spectroscopy (RS) in association with partial least squared discriminant analysis (PLS-DA).

Sterility testing is a crucial step in quality control of pharmaceutical drug products before their commercial release. However, the current procedures for bioburden testing, which are primarily growth based, are highly time-consuming, costly and have limited sensitivity and specificity.

RS is under investigation as a lower cost, more rapid alternative; however, researchers have struggled to balance robustness, sensitivity, cost and a low limit of detection (LOD).

According to a new study, currently available in pre-print at bioRxiv, key challenges that new techniques must overcome include:

(a) discrimination between Raman spectra from organic molecules in the formula and bacterial ones
(b) detection at low contamination, overcoming the weak signal from bacteria
(c) contribution to the Raman signal from other sources such as product packaging, fluorescent compounds
(d) correct data processing and statistical analysis model.

The study presents an approach to up-concentrate and detect ≤10 colony forming units (CFU)/ml of relevant bacteria with RS and multivariate analysis without breaching the primary drug product package.

To increase the Raman signal and the likelihood contaminants would be detected, the vials were centrifuged in an inclined (upside-down) position to localize the bacteria close to the neck of the product vial.

The RS-PLS-DA approach enabled the fast and non-invasive discrimination of products vials containing low numbers of bacteria from sterile ones without breaching the packaging. The technique enabled the identification of three different bacteria Bacillus subtilis, Salmonella enterica and Staphylococcus haemolyticus, as well as B. subtilis spores with an accuracy of 99 percent. The method was able to distinguish samples with vegetative cells and spores in limits <10 CFU/ml, even in the presence of other organic molecules from the product formula in the container.

Independent validation was able to confirm the high sensitivity and specificity.

According to Grosso et al., the project supports the use of the RS-PLS-DA approach as alternative to the pharmacopeial destructive sterility testing method. “We provide a feasible approach using RS in association with PLS-DA to detect extremely low numbers of cells or spores with high accuracy and reproducibility without compromising the robustness of the method,” wrote the authors. “These results support Raman spectroscopy as a promising biotechnological tool suitable for bioburden test in quality control of pharmaceutical industry.”

They added that the RS-PLS-DA method developed could enable real-time monitoring of contamination in pharmaceutical processing.