Thursday, September 1, 2022

Using Organic Metal Nanohybrid Structures to Simultaneously Identify Multiple Food Poisoning Bacteria

Osaka Metropolitan University scientists have developed a simple, rapid method to simultaneously identify multiple food poisoning bacteria, based on color differences in the scattered light by nanometer-scaled organic metal nanohybrid structures (NHs) that bind via antibodies to those bacteria. This method is a promising tool for rapidly detecting bacteria at food manufacturing sites and thereby improving food safety. The findings were published in Analytical Chemistry.

[Image description: Introducing antibodies that specifically bind to bacteria into nanometer-scaled hybrid structures of polymer-coated metal nanoparticles and then using these structures as test labels, OMU scientists successfully detected food poisoning bacteria E. coli O26, E. coli O157, and S. aureus as white, red, and blue scattered light under the microscope.]

According to the World Health Organization (WHO), every year food poisoning affects 600 million people worldwide—almost 1 in every 10 people—of which 420,000 die. Bacterial tests are conducted to detect food poisoning bacteria at food manufacturing factories, but it takes more than 48 hours to obtain results due to the time required for a bacteria incubation process called culturing. Therefore, there remains a demand for rapid testing methods to eliminate food poisoning accidents.

Responding to this need, the research team led by Professor Hiroshi Shiigi at the Graduate School of Engineering, Osaka Metropolitan University, utilized the optical properties of organic metal NHs—composites consisting of polyaniline particles that encapsulate a large number of metal nanoparticles—to rapidly and simultaneously identify food poisoning-inducing bacteria called enterohemorrhagic Escherichia coli (E. coli O26 and E. coli O157) and Staphylococcus aureus.

The team first found that organic metal NHs produced stronger scattered light than metal nanoparticles of the same size. Since the scattered light of these NHs is stable in the air for a long period of time, they are expected to function as stable and highly sensitive labeling materials. Furthermore, it has been revealed that these NHs exhibit different colors of scattered light (white, red, and blue) depending on the metal elements of the nanoparticles (gold, silver, and copper).

Then the team introduced antibodies that bind specifically to E. coli O26, E. coli O157, and S. aureus into the organic metal NHs and used these NHs as labels to evaluate the binding properties of the antibody-conjugated NHs to specific bacterial species. As a result, E. coli O26, E. coli O157, and S. aureus were observed as white, red, and blue scattered light, respectively, under the microscope. Furthermore, when adding predetermined amounts of E. coli O26, E. coli O157, and S. aureus to rotten meat samples containing various species of bacteria, the team succeeded in using these labels to simultaneously identify each bacterial species added.

This method can identify various types of bacteria by changing the antibodies to be introduced. In addition, since it does not require culturing, bacteria can be rapidly detected within one hour, increasing its practicality as a new testing method.

Professor Shiigi commented, “We aim to establish new detection principles and testing methods through the development of unique nano-biomaterials. Through this development, we hope to contribute not only to food safety and security, but also to the formation of a safe and affluent society in terms of stable supply and quality control of functional foods, medical care, drug discovery, and public health.”

Funding

This work was financially supported by a JST START Grant (Number JPMJST1916). The authors also acknowledge financial support from the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (A) (KAKENHI 21H04963).

Paper Information

Simultaneous Optical Detection of Multiple Bacterial Species Using Nanometer- Scaled Metal−Organic Hybrids. Analytical Chemistry. Author: So Tanabe, Satohiro Itagaki, Kyohei Matsui, Shigeki Nishii, Yojiro Yamamoto, Yasuhiro Sadanaga, Hiroshi Shiigi. 

Source: Osaka Metropolitan University

Thursday, August 25, 2022

Final Revision to Annex 1 Published - Rapid Methods Highlighted

After many years, the final revision to Annex 1 "Manufacture of Sterile Medicinal Products," under the The Rules Governing Medicinal Products in the European Union Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use, was published today, and will go into effect August 25, 2023.

The revision may be downloaded here: https://health.ec.europa.eu/system/files/2022-08/20220825_gmp-an1_en_0.pdf.

Of note, the use of rapid and alternative microbiological methods is encouraged. Specifically:

"The use of appropriate technologies (e.g. Restricted Access Barriers Systems (RABS), isolators, robotic systems, rapid/alternative methods and continuous monitoring systems) should be considered to increase the protection of the product from potential extraneous sources of endotoxin/pyrogen, particulate and microbial contamination such as personnel, materials and the surrounding environment, and assist in the rapid detection of potential contaminants in the environment and the product."

"The adoption of suitable alternative monitoring systems such as rapid methods should be considered by manufacturers in order to expedite the detection of microbiological contamination issues and to reduce the risk to product. These rapid and automated microbial monitoring methods may be adopted after validation has demonstrated their equivalency or superiority to the established methods."

"For products with short shelf life, the environmental data for the time of manufacture may not be available; in these cases, the compliance should include a review of the most recent available data. Manufacturers of these products should consider the use of rapid/alternative methods."

Some of the most encouraging changes are associated with the use of rapid methods instead of the standard methods for environmental monitoring. For example, one of the notes in Table 2, which discusses the maximum permitted microbial contamination level during qualification of a cleanroom, states, "Limits are applied using CFU throughout the document. If different or new technologies are used that present results in a manner different from CFU, the manufacturer should scientifically justify the limits applied and where possible correlate them to CFU." Therefore, the EU allows us to revise the numerical values for the CFU-based maximum permitted microbial contamination level to new numerical values based on a rapid method's alternative microbial detection signal. One example of a new rapid signal is an intrinsic fluorescent count which can be derived in real-time and does not require growth on conventional microbiological media. 

Similar guidance on the use of alternative limits is provided in the footnotes for Table 6, which discusses the maximum action limits for viable particle contamination during routine manufacturing. Additionally, although the table provides CFU-based limits for active air samples, settle plates, contact plates and glove prints, one footnote allows for the use of methods other than those specified in the table. Specifically, "[I]t should be noted that the types of monitoring methods listed in the table above are examples and other methods can be used provided they meet the intent of providing information across the whole of the critical process where product may be contaminated (e.g. aseptic line set-up, aseptic processing, filling and lyophilizer loading)." 

The finalization of this long-awaited revision is welcomed and demonstrates  the EU's commitment to advancing the detection of microorganisms during sterile drug manufacturing by encouraging and implementing 21st Century rapid microbiological methods.

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.

Tuesday, June 7, 2022

New Blood Test Can Help Doctors Diagnose Tuberculosis and Monitor Treatment

Researchers at Tulane University School of Medicine have developed a new highly sensitive blood test for tuberculosis (TB) that screens for DNA fragments of the Mycobacterium tuberculosis bacteria that causes the deadly disease.

The test could give doctors a new tool to both quickly identify TB and then gauge whether drug treatments are effective by monitoring levels of DNA from the pathogen circulating through the bloodstream, according to a new study published in the journal The Lancet Microbe.

Tuberculosis is now the second most deadly infectious disease in the world, behind only COVID-19. In 2020, an estimated 10 million people contracted TB and 1.5 million people died from it, according to the World Health Organization. 

Most TB tests rely on screening sputum, a thick type of mucus from the lungs. But collecting sputum from patients suspected of having TB can be difficult, especially for children. TB can also be harder to diagnose in immunocompromised HIV patients and others where the infection migrates outside of the lungs into other areas of the body. In these extrapulmonary cases, patients can have little bacteria in the sputum, which leads to false negatives using current testing methods, said lead study author Tony Hu, PhD, Weatherhead Presidential Chair in Biotechnology Innovation at Tulane University.

“This assay may be a game-changer for TB diagnoses that not only provides accurate diagnosis results but also has the potential to predict disease progression and monitor treatment,” Hu said. “This will help doctors rapidly intervene in treatment and reduce the risk of death, especially for children living with HIV.”

The study evaluated a CRISPR-based assay that screened for cell-free DNA from live Mycobacterium tuberculosis bacilli. The screening target is released into the bloodstream and cleared quite rapidly, providing a real-time snapshot of active infection.

Researchers tested preserved blood samples from 73 adults and children with presumptive TB and their asymptomatic household contacts in Eswatini, Africa. 

The test identified adult TB with 96.4% sensitivity and 94.1% specificity and pediatric TB with 83.3% sensitivity and 95.5% specificity. (Sensitivity refers to how well a test can diagnose a positive case, while specificity is a measure of a test’s accurately determining a negative case.)

Researchers also tested 153 blood samples from a cohort of hospitalized children in Kenya. These were HIV-positive patients who were at high risk for TB and presented with at least one symptom of the disease. The new test picked up all 13 confirmed TB cases and almost 85% of unconfirmed cases, which were cases that were diagnosed due to clinical symptoms and not existing gold standard testing methods.

The CRISPR-based test uses a small blood sample and can deliver results within two hours.

“We are particularly excited that the level of Mycobacterium tuberculosis cell-free DNA in HIV-infected children began to decline within a month of treatment, and most of the children's blood was cleared of the bacteria DNA fragments after treatment, which means that CRISPR-TB has the potential to monitor treatment and will give physicians the ability to better treat worldwide TB infections,” Hu said.

The researchers have since adapted the assay to a rapid test platform that can deliver results in 30 minutes without any special equipment. Results would be viewable on a paper strip like a rapid COVID-19 test.  

“A highly accurate, rapid blood test that could be used anywhere would benefit millions of people living in resource-limited areas with a high TB burden,” Hu said.

The full results of the paper are online here

Source: Tulane University School of Medicine 

Portable Sensor Technology Aims to Quickly Detect Foodborne Contaminants Outside the Lab

An international team led by a University of Massachusetts Amherst food and environmental virologist has received a $750,000 USDA National Institute of Food and Agriculture (NIFA) partnership grant to develop and test portable, rapid biosensors capable of detecting noroviruses and mycotoxins in foods and agricultural products. It is among the first partnership grants awarded with an international partner by the USDA.

Noroviruses are the leading cause of foodborne illness globally, and are highly contagious, causing pandemics every few years, says lead investigator Matthew Moore, assistant professor of food science. Moore will work with UMass Amherst food science colleague John Gibbons, a fungi expert, and food science Ph.D. candidate Sloane Stoufer in the Moore Lab. The UMass team will collaborate with senior lecturer and principal investigator Marloes Peeters and postdoctoral research associate Jake McClements at Newcastle University’s School of Engineering in England.

“People can get really sick from foods that contain viruses and toxins,” Moore says. “We need a way to quickly and easily find out if a food contains these contaminants in a cheap but effective way – without the need to go back to a separate lab to do the testing.”

Mycotoxins are toxic substances produced by fungi that can grow in warm and humid conditions on crops and food, in particular in many grains, produce, nuts, seeds and spices. They represent a growing threat to public health in the face of climate change trends and increased consumption of plant-based foods, Moore says.

“One of the interesting things about mycotoxins as a foodborne contaminant is that they’re often not very acute, so you’re less likely to notice it,” Moore says. “Oftentimes, the damage they do is more chronic, and they will mess with the kidneys and liver especially and can promote cancer.”

That makes early detection all the more important. “With this technology we’re trying to create a cheap, highly durable, and potentially reusable sensor that can detect these contaminants,” Moore says.

The UMass Amherst food scientists got together with engineers at Newcastle University to seek a rare international partnership grant from the USDA’s NIFA. The British engineers are world leaders in electrochemical sensing techniques based on generating molecularly imprinted polymer nanoparticles (nanoMIPs).

“The grant enables an unprecedented international exchange,” Moore says. The UMass team will learn more about the application of nanoMIPs when they visit the Peeters Lab at Newcastle, and the UK team will be hosted by Moore’s Applied and Environmental Virology Lab to gain knowledge about virological, microbiological and food science techniques.

“This nanoMIP-based sensing technology has numerous advantages,” Moore says. “It is very stable in intense conditions, and very portable. It is also quite inexpensive, a very important consideration in testing for foods.”

NanoMIP-based electrochemical sensing is an exciting new application for agricultural targets. “The technology has already shown promise for other targets, including SARS-CoV-2, and we hope to further explore its potential for human noroviruses and mycotoxins,” Moore says.

Source: University of Massachusetts Amherst 

Saturday, May 14, 2022

New Smartphone-Powered Microchip Opens the Door for Faster, Cheaper At-Home Medical Testing

A University of Minnesota Twin Cities research team has developed a new microfluidic chip for diagnosing diseases that uses a minimal number of components and can be powered wirelessly by a smartphone. The innovation opens the door for faster and more affordable at-home medical testing.

The researchers' paper is published in Nature Communications, a peer-reviewed, open access, scientific journal published by Nature Research. Researchers are also working to commercialize the technology.

Microfluidics involves the study and manipulation of liquids at a very small scale. One of the most popular applications in the field is developing "lab-on-a-chip" technology, or the ability to create devices that can diagnose diseases from a very small biological sample, blood or urine, for example.

Scientists already have portable devices for diagnosing some conditions-;rapid COVID-19 antigen tests, for one. However, a big roadblock to engineering more sophisticated diagnostic chips that could, for example, identify the specific strain of COVID-19 or measure biomarkers like glucose or cholesterol, is the fact that they need so many moving parts.

Chips like these would require materials to seal the liquid inside, pumps and tubing to manipulate the liquid, and wires to activate those pumps-;all materials that are difficult to scale down to the micro level. Researchers at the University of Minnesota Twin Cities were able to create a microfluidic device that functions without all of those bulky components.

Many lab-on-a-chip technologies work by moving liquid droplets across a microchip to detect the virus pathogens or bacteria inside the sample. The University of Minnesota researchers' solution was inspired by a peculiar real-world phenomenon with which wine drinkers will be familiar-;the "legs," or long droplets that form inside a wine bottle due to surface tension caused by the evaporation of alcohol.

Using a technique pioneered by Oh's lab in the early 2010s, the researchers placed tiny electrodes very close together on a 2 cm by 2 cm chip, which generate strong electric fields that pull droplets across the chip and create a similar "leg" of liquid to detect the molecules within.

Because the electrodes are placed so closely together (with only 10 nanometers of space between), the resulting electric field is so strong that the chip only needs less than a volt of electricity to function. This incredibly low voltage required allowed the researchers to activate the diagnostic chip using near-field communication signals from a smartphone, the same technology used for contactless payment in stores.

This is the first time researchers have been able to use a smartphone to wirelessly activate narrow channels without microfluidic structures, paving the way for cheaper, more accessible at-home diagnostic devices.

"This is a very exciting, new concept," said Christopher Ertsgaard, lead author of the study and a recent CSE alumnus (ECE Ph.D. '20). "During this pandemic, I think everyone has realized the importance of at-home, rapid, point-of-care diagnostics. And there are technologies available, but we need faster and more sensitive techniques. With scaling and high-density manufacturing, we can bring these sophisticated technologies to at-home diagnostics at a more affordable cost."

Oh's lab is working with Minnesota startup company GRIP Molecular Technologies, which manufactures at-home diagnostic devices, to commercialize the microchip platform. The chip is designed to have broad applications for detecting viruses, pathogens, bacteria, and other biomarkers in liquid samples.

"To be commercially successful, in-home diagnostics must be low-cost and easy-to-use," said Bruce Batten, founder and president of GRIP Molecular Technologies. "Low voltage fluid movement, such as what Professor Oh's team has achieved, enables us to meet both of those requirements. GRIP has had the good fortune to collaborate with the University of Minnesota on the development of our technology platform. Linking basic and translational research is crucial to developing a pipeline of innovative, transformational products."

In addition to Oh and Ertsgaard, the research team included University of Minnesota Department of Electrical and Computer Engineering alumni Daniel Klemme (Ph.D. '19) and Daehan Yoo (Ph.D. '16) and Ph.D. student Peter Christenson.

This research was supported by the National Science Foundation (NSF). Oh received support from the Sanford P. Bordeau Endowed Chair at the University of Minnesota and the McKnight University Professorship. Device fabrication was performed in the Minnesota Nano Center at the University of Minnesota, which is supported by NSF through the National Nanotechnology Coordinated Infrastructure (NNCI).

Source: University of Minnesota

Journal reference: Ertsgaard, C. T., et al. (2022) Open-channel microfluidics via resonant wireless power transfer. Nature Communications.  doi.org/10.1038/s41467-022-29405-2.

New Rapid Virus Test uses Gold Particles and is 150 Times More Accurate than Standard Tests

University of Texas at Dallas researchers have developed a rapid virus test using gold particles and lasers that promises to deliver results as accurate as lab tests in a fraction of the time.

The technology, called digital plasmonic nanobubble detection—or Diamond for short—is 150 times more accurate than standard rapid tests, according to a study published in Nature Communications last month. Its accuracy is comparable to polymerase chain reaction (PCR) tests, which take hours to perform.

The team of UTD scientists that authored the study, led by associate professor of mechanical engineering Dr. Zhenpeng Qin, tested Diamond against respiratory syncytial virus, although the researchers say the technology can be used to detect other prominent viruses, like COVID-19 and influenza.

"For the [PCR] COVID test, we drive through the pharmacy and give the sample. Getting the sample tested usually takes two to four hours before we get the results," said Haihang Ye, a UTD research associate in mechanical engineering. "Our technology can reduce the sample testing time to 30 minutes, but the sensitivity can be as good as those molecular tests."

Faster, cheaper and more effective virus tests are in high demand as the U.S. shifts into a new normal in the coronavirus pandemic. Though case counts are near all-time lows in North Texas, the highly contagious BA.2 variant continues to spread locally and across the country.

The cost of a COVID-19 test varies widely depending on location and type of test. A study of the largest hospitals in every state done by the Peterson Center of Healthcare and Kaiser Family Foundation Health System Tracker found coronavirus test prices ranged anywhere from $20 to more than $1,400. Only 3% of the hospitals surveyed listed testing prices below $50.

A Diamond test, which Ye said can be produced for around $15, mixes a patient sample from a nasal swab with gold nanoparticles attached to antibodies for the virus being tested. The antibodies, marked by the gold nanoparticles, then bind with proteins on the virus' surface if the virus is present in the sample.

Researchers then inject the sample mixed with labeled antibodies into a narrow tube mounted on a glass slide. As the liquid passes through the tube, it's hit by the beams of two lasers, one of which activates the gold nanoparticles, causing them to expand.

If the expansion is strong enough, the nanoparticle will boil the water around it and create vapor bubbles. Large nanobubbles mean the virus is present in the sample.

"If there's no virus, there will be a tiny nanobubble signal from the particle only so we can differentiate the sample's status," said Yaning Liu, a UTD mechanical engineering doctoral student and co-first author of the Diamond study.



Diamond is the product of years of research and millions of dollars in grant funding, including $2.5 million in grants from the National Institute of Allergy and Infectious Diseases and a $293,000 grant from the Department of Defense's Congressionally Directed Medical Research Programs.

To test different viruses using the technology, all researchers need to do is change the associated antibodies, Ye said. Though Diamond has the potential to expand testing options for a number of viruses, it requires researchers to know what they're testing for.

"One of the challenges with the current tests is that providers have to kind of have an idea of what they're looking for," said Elitza Theel, associate professor of laboratory medicine and pathology at the Mayo Clinic in Rochester, Minn.

A less-targeted approach using a technology called metagenomic next-generation sequencing allows scientists to sequence all of the genetic material in a sample to identify which infectious pathogens are present. The technology is already in use, but the process is expensive and takes days to return results, Theel said.

"It's not really helpful in the immediate acute setting," she said.

While Diamond must be approved by the Food and Drug Administration before it can be used publicly, the scientists behind the technology launched a company called Avsana Labs to hopefully commercialize it. Qin serves as president of the company, which was created through UTD's Venture Development Center.

Just last week, another North Texas company had its COVID-19 test approved by the FDA. Frisco-based InspectIR Systems invented a breathalyzer apparatus, the first coronavirus test of its kind to get federal approval, that can yield results in less than three minutes.

Yaning Liu et al, Digital plasmonic nanobubble detection for rapid and ultrasensitive virus diagnostics, Nature Communications (2022). DOI: 10.1038/s41467-022-29025-w

Rapid Method Shown to Detect Infection in Cystic Fibrosis

Rapid method shown to detect infection in cystic fibrosis: Southampton researchers have demonstrated a quick and accurate method to diagnose bacterial infections. The technique has the potential to detect infections in cystic fibrosis patients in minutes rather than days.

In future, the simple analysis could be performed on hospital wards to deliver faster and more effective treatment.

The approach could also be expanded to target a variety of diseases and counter anti-microbial resistance.

Cystic fibrosis is an inherited condition that causes sticky mucus to build up in the lungs and digestive system. This causes lung infections and problems with digesting food.

It affects around 1 in every 10,000 births in the UK.

Treatments are available to help reduce the problems caused by the condition. Yet recurring infections still dramatically reduce the quality and length of life.

The current methods for diagnosing immediate (acute) and longer-term (chronic) infections are complex and time-consuming in the laboratory. For biofilm infections, it can take days from collecting and processing a patient’s sample to achieving a result. This delays effective treatments and impacts patient outcomes.

A multi-disciplinary team from the University of Southampton and University Hospital Southampton set out to develop a diagnostic tool that would be rapid, accurate and simple-to-use for doctors.

They have developed a new chemical analysis technique called multi-excitation Raman spectroscopy. This non-invasive method emits a scattering of multiple colours of light into a patient’s sample.

Prof Sumeet Mahajan, Head of Chemical Biology and the Associate Director of Institute for Life Sciences at the University of Southampton, explained:

“When light is applied to a sample’s molecules they can vibrate which helps us understand their characteristics. By using different colours of light, a different set of such vibrations can be triggered meaning we can get more information about their composition than previously possible.

“This then allows ‘finger-printing’ that can be used to identify the properties of the pathogens that cause cystic fibrosis. In many current techniques, a reagent needs to be added to a sample or a tag needs to be attached to the molecules of interest to analyse their composition. This is not required under this new approach which uses natural properties of the molecules to analyse them.”

Professor Mahajan continued: “Our new Raman spectroscopy based method offers many advantages over resource-intensive, culture-based methods, allowing rapid and label-free analysis. It is reagentless and avoids complex sample-preparation steps with sophisticated equipment. Here, we have developed a method that is highly accurate yet rapid and neither requires nanoscale materials for enhancing signals nor fluorophores for detection.”

Long term infections in the lungs of people with cystic fibrosis are extremely hard to treat . There is evidence that the Pseudomonas aeruginosa bacteria exists as biofilms in the body, protecting the bacteria from antibiotic action and driving antimicrobial resistance. This increases the urgency for rapid and effective treatment.

The Southampton research, published in Analytical Chemistry, showed 99.75% accuracy at identifying Pseudomonas aeruginosa and Staphylococcus aureus across all studied strains. This included 100% accuracy for drug-sensitive and drug-resistant Staphylococcus aureus.

The project drew together expertise from the National Institute for Health and Care Research (NIHR) Southampton Biomedical Research Centre (BRC) and Southampton Clinical Research Facility (CRF), the National Biofilms Innovation Centre (NBIC), together with the University of Southampton’s School of Chemistry and Institute for Life Sciences (IfLS). It was led by Professor Mahajan, Professor Jeremy Webb and Professor Saul Faust.

Prof Faust, Director of NIHR Southampton CRF, said: “Our study demonstrates an important step toward a rapid and reagentless diagnostic tool requiring only simple or routine sample preparation.

“Such a platform could also prove useful in a variety of other disease areas and help address the mounting challenge of anti-microbial resistance.”

Saturday, March 5, 2022

'Fingerprint' Machine Learning Technique Identifies Different Bacteria in Seconds​

Bacterial identification can take hours and sometimes longer, valuable time when diagnosing infections and choosing acceptable therapies. There could also be a faster, extra correct course of in response to researchers at KAIST. By educating a deep studying algorithm to establish the “fingerprint” spectra of the molecular parts of assorted micro organism, the researchers might classify numerous micro organism in several media with accuracies of as much as 98%.

Their outcomes have been made obtainable on-line on Jan. 18 in Biosensors and Bioelectronics, forward of publication within the journal’s April subject.

Micro organism-induced sicknesses, these brought on by direct bacterial an infection or by publicity to bacterial toxins, can induce painful signs and even result in dying, so the speedy detection of micro organism is essential to stop the consumption of contaminated meals and to diagnose infections from scientific samples, akin to urine. “By utilizing surface-enhanced Raman spectroscopy (SERS) evaluation boosted with a newly proposed deep learning model, we demonstrated a markedly easy, quick, and efficient path to classify the indicators of two widespread micro organism and their resident media with none separation procedures,” mentioned Professor Sungho Jo from the College of Computing.

Raman spectroscopy sends gentle by way of a pattern to see the way it scatters. The outcomes reveal structural details about the pattern—the spectral fingerprint—permitting researchers to establish its molecules. The surface-enhanced model locations pattern cells on noble steel nanostructures that assist amplify the pattern’s indicators.

Nevertheless, it’s difficult to acquire constant and clear spectra of micro organism on account of quite a few overlapping peak sources, akin to proteins in cell partitions. “Furthermore, sturdy indicators of surrounding media are additionally enhanced to overwhelm goal indicators, requiring time-consuming and tedious bacterial separation steps,” mentioned Professor Yeon Sik Jung from the Division of Supplies Science and Engineering.

To parse by way of the noisy indicators, the researchers applied a synthetic intelligence technique known as deep studying that may hierarchically extract sure options of the spectral info to categorise information. They particularly designed their mannequin, named the dual-branch wide-kernel community (DualWKNet), to effectively be taught the correlation between spectral options. Such a capability is crucial for analyzing one-dimensional spectral information, in response to Professor Jo.

Schematics of the general process of Raman data collection and analysis where a single spectrum is attained from a single cell and classified via deep learning

“Regardless of having interfering indicators or noise from the media, which make the overall shapes of various bacterial spectra and their residing media indicators look comparable, excessive classification accuracies of bacterial sorts and their media have been achieved,” Professor Jo mentioned, explaining that DualWKNet allowed the crew to establish key peaks in every class that have been nearly indiscernible in particular person spectra, enhancing the classification accuracies. “In the end, with the usage of DualWKNet changing the micro organism and media separation steps, our technique dramatically reduces evaluation time.”

The researchers plan to make use of their platform to review extra micro organism and media sorts, utilizing the knowledge to construct a coaching information library of assorted bacterial sorts in extra media to cut back the gathering and detection instances for brand new samples.

“We developed a significant common platform for speedy bacterial detection with the collaboration between SERS and deep studying,” Professor Jo mentioned. “We hope to increase the usage of our deep learning-based SERS evaluation platform to detect quite a few varieties of bacteria in extra media which can be necessary for meals or scientific evaluation, akin to blood.”

Reference

Eojin Rho et al, Separation-free bacterial identification in arbitrary media by way of deep neural network-based SERS evaluation, Biosensors and Bioelectronics (2022). 

Sunday, February 6, 2022

Simple, Inexpensive, Fast and Accurate Nano-sensors Pinpoint Infectious Diseases

A very interesting paper has been published by Arizona State University scientists that describes a design for an in-solution assay pipeline, featuring nanobody-functionalized nanoparticles for rapid, electronic detection (Nano2RED) of Ebola and COVID-19 antigens. Without requiring any fluorescent labelling, washing, or enzymatic amplification, these multivalent AuNP sensors reliably transduce antigen binding signals upon mixing into physical AuNP aggregation and sedimentation processes, displaying antigen-dependent optical extinction readily detectable by spectrometry or portable electronic circuitry.

The university has summarized the research in a review article from its news department, and is replicated below.

In recent years, deadly infectious diseases, including Ebola and COVID-19, have emerged to cause widespread human devastation. Although researchers have developed a range of sophisticated methods to detect such infections, existing diagnostics face many limitations.

In a new study, Chao Wang, a researcher at Arizona State University’s Biodesign Institute and School of Electrical, Computer & Energy Engineering, along with ASU colleagues and collaborators at the University of Washington (UW), Seattle describe a novel method for detecting viruses like Ebola virus (EBOV) and SARS CoV-2.

The technique, known as Nano2RED, is a clever twist on conventional high-accuracy tests relying on complex testing protocols and expensive readout systems. The in-solution nano-sensors (“Nano2” in the name) serve to detect disease antigens in a sample by simple mixing. The innovative Rapid and Electronic Readout process (“RED”) developed in the Wang lab delivers test results, which are detectable as a color change in the sample solution, and record the data through inexpensive semiconductor elements such as LEDs and photodetectors.

The technology represents a significant advance in the fight against infectious diseases. It can be developed and produced at very low cost, deployed within weeks or days after an outbreak, and made available for around 1 cent per test.

Compared with widely used high-accuracy lab tests, such as ELISA, Nano2RED is much easier to use. It does not require surface incubation or washing, dye labelling, or amplification, yet still provides about 10 times better sensitivity than ELISA. In addition, the use of semiconductor devices supports a highly portable digital readout system, which can be developed and produced at a cost as low as a few dollars, making it ideal not only for lab use but for clinics, home use, and remote or resource-strained locations. This approach is based on modular designs, and could potentially be used to test for any pathogen.

“This technology works not because it is complex but because it is simple,” says professor Wang. “Another unique feature is the multidisciplinary nature of biosensing. A fundamental understanding of biochemistry, fluidics, and optoelectronics helped us come up with something this ‘simple’.”

Wang is a researcher with the Biodesign Center for Molecular Design and Biomimetics at ASU. He is also a researcher with ASU’s School of Electrical, Computer and Energy Engineering; and the Center for Photonic Innovation. Dr. Liangcai Gu is the collaborator at Department of Biochemistry and Institute for Protein Design at UW, Seattle.

The research appears in the current issue of the journal Biosensors and Bioelectronics. Dr. Xiahui Chen and Md Ashif Ikbal from ASU and Dr. Shoukai Kang from UW are the first authors, and Jiawei Zuo and Yuxin Pan are the other contributing authors.

The testing bottleneck

Epidemiologists have long known the basic formula when confronting a disease outbreak. To identify cases and stop the contagion, it is necessary to develop an accurate test or assay that can identify the disease, then test early and often, to assess the rate of spread and attempt to isolate the infected.

Unfortunately, by the time a new diagnostic has been developed, manufactured, and distributed, the disease outbreak is often already widespread and challenging to contain. Further, accurate tests including PCR, (which can amplify tiny levels of pathogenic nucleic acids to measurable levels), are often expensive, labor-intensive and require sophisticated laboratory facilities.

The Ebola epidemic of 2014-2016, though largely confined to West Africa, spread with terrifying speed, causing panic and killing more than 11,000 people. The virus’ rate of lethality, one of the highest for any known pathogen, can exceed 90%, depending on disease strain. The crisis was exacerbated by a combination of inadequate surveillance systems and poor public health infrastructure.

SARS CoV-2, though less lethal than Ebola, has spread to every country on earth and has already killed more than 5.6 million people worldwide. In both disease outbreaks, diagnostic testing arrived late on the scene. Further, costly and cumbersome testing requirements have meant that far too few tests have been administered, even after their successful development.

The new study applies its innovative method to test for these two prominent diseases, as a proof of concept.

Tidal wave

A common feature in many disease outbreaks is the lightning speed with which a pathogen, having first infected a handful of people, can gather momentum, fan out in all directions and quickly overwhelm hospitals and healthcare providers. Cutting off a pathogen’s routes of transmission requires identifying and isolating sick individuals through testing, as quickly as possible.

During a pandemic like COVID-19, the sensitivity of a given diagnostic test is secondary to how often the test is given and how long it takes for results to be processed. A highly sensitive test is of limited use if it can only be given once, and results require a weeks-long turnaround. Research has shown that infection outbreaks are best controlled when testing is repeated in less than 3-day intervals and at a large scale.

Adequately preparing society for current and future outbreaks of infectious disease will require faster, cheaper, more accurate and more easily usable diagnostics.

Close affinity

The new technology can identify secreted glycoprotein (sGP), a telltale fingerprint of Ebola virus disease and the SARS-CoV-2 spike protein receptor binding domain (RBD). The technology is highly accurate, rivaling ELISA, a long-recognized gold standard technology for diagnostic testing.

The basic idea of such diagnostics, known as immunoassays, is simple: a sample of blood (or other biological fluid) is applied to the assay, which is adorned with antibodies. When antibodies recognize the presence of a corresponding disease antigen in the sample, they bind with it, producing a positive test result. In the ELISA test, the disease antigen needs to be immobilized on a flat surface.

Nano2RED also relies on binding affinity for positive diagnosis but instead uses floating gold nanoparticles for readout. Unlike ELISA, Nano2RED can be developed from scratch in roughly 10 days and theoretically applicable for any pathogen, providing vitally important early surveillance in the case of a disease outbreak. It can deliver test results in 15-20 minutes and may be administered at an estimated cost of a penny per test. In the current study, the new test was shown to detect Ebola’s sGP in serum with a sensitivity roughly 10 times better than ELISA.

Sensing danger

The accompanying graphic shows how the method works: The first step is to produce a very large library containing over a billion random amino acid sequences known as nanobodies, that can act as synthetic antibodies, able to bind with target disease antigens. This vast library of nanobodies is then successively screened against the antigen in question, for example sGP in the case of Ebola. Only those nanobodies that show strong binding affinity for the antigen are used for sensing.


Next, the selected nanobodies are affixed to gold nanoparticles that will act as probes to identify and bind with antigens present in a blood sample. In the study, a pair of two high affinity nanobodies were selected and attached to gold nanoparticles. This approach improves both the sensitivity and specificity of the test.

The key innovation of Nano2RED is the way the antigen detection is registered. As disease antigens in the sample are recognized by the nanobodies, they bind together, forming clusters of bound nanobody and antigen, like islands of algae floating on the sea surface. “Basically, an antigen works like superglue to bring the nanoparticle together,” Wang says. 

A “golden” opportunity in the fight against infectious disease

The gold nanoparticles provide a stable platform to hold the nanobodies in place.  Once enough binding has taken place, the bound clusters begin to sink to the bottom of the vessel. This can be detected with the naked eye in the form of a color change. The solution becomes lighter in color as the gold-nanoparticle-carried antigen-antibody clusters precipitate out, signaling detection of the pathogen.

While full precipitation of nanoparticle clusters leading to a test result can normally take several hours, the process can be sped up by centrifuging the sample, which eliminates the wait time for precipitation. In this case, just 15-20 minutes are sufficient for a result. “Of course, the gold nanoparticles are heavy, and that helps quick sedimentation, too.” Wang added.

Gold nanoparticles also work to display color, not yellow but red here, by absorbing light from a narrow spectral range. This absorbance feature allowed the Wang lab to invent  a tiny, inexpensive device that converts this color change into an electrical signal, using color-matching semiconductor LEDs and photodetectors. Such instruments produce a rapid and accurate readout of assay results, whose limits of detection are comparable to or better than costly lab-based spectroscopy methods.

The test also delivers quantitative results based on amount of antigen detected. This could be vitally useful for estimating disease severity as well as time elapsed since the infection event. In the future, the test results can be digitized by circuits and conveniently transmitted via internet to anywhere in the world for data analysis and further scrutiny, which could be important to government policy decision-making processes and timely interruption of the transmission.

Nano2RED requires only a tiny blood sample, typically around 20 microliters. “This also makes gold inexpensive in our case, because the mount we need is so tiny.” Wang said. Unlike conventional methods, Nano2RED is also very simple to use with minimal training involved for healthcare personnel. It does not require any time-consuming and expensive incubation, washing, fluorescent labeling or amplification.

Future research will help improve the assay’s limits of detection even further and modify its detection capacities to include virion particles, extracellular vesicles, small molecules, and nucleic acids. “There is certainly still a lot to explore” Wang added, “but we so far have a happy marriage between engineering disciplines and biology. And that is what we will continue to work on.”

This research was supported by the National Science Foundation and National Institutes of Health.

Source: Arizona State University