Saturday, February 4, 2023

New Electrochemical Sensing Technology Enables Detection of SARS-CoV-2 Antigen-specific Antibodies

Not all SARS-CoV-2 infections are created equal. We have learned this through multiple virus waves are taking their toll on the world's population. Improving vaccines and new anti-viral therapies that target distinct viral molecules (antigens) and the changes they undergo over time have helped to soften this blow. However, to control the disease even better and everywhere, we have to be able to assess whether and with which viral variant individuals have been infected, what kind of protective immunity they possess, and how they respond to vaccinations and therapies.

An obvious way to accomplish this is through the detection of antibodies that the immune system produces against the virus' proteins and variant-specific antigens. Importantly, currently available COVID vaccines induce the production of antibodies against the Spike (S) protein, but rarely the N protein, while natural infection produces antibodies against both proteins. This allows the immune responses to be clearly distinguished from each other. Having a way to detect these different antibody types could inform health care and drug development decisions in a more systematic way. However, current antibody detection technologies are time-consuming, too costly, often require clinical laboratories, are and not able to accurately measure the levels of antibodies against multiple antigens, or they suffer from a combination of these inadequacies – which prevent them from being able to rapidly and effectively generate data about antibodies across global populations.

Now, an in-depth study from a research team at the Wyss Institute for Biologically Inspired Engineering at Harvard University demonstrates that the Institute's portable electrochemical sensing technology known as eRapid could be an ideal instrument to enable the inexpensive, multiplexed detection of different SARS-CoV-2-directed antibodies at the point-of-care. The team, led by Wyss Founding Director Donald Ingber, M.D., Ph.D. and Wyss Senior Scientist Pawan Jolly, Ph.D., showed that specifically engineered eRapid sensors can detect antibodies targeting the virus' so-called nucleocapsid (N) protein from ultra-small samples of blood plasma and dried blood spots with 100% sensitivity and specificity within less than 10 minutes. The findings are published in Biosensors and Bioelectronics.

Taking aim at COVID-induced immunity

"The study's findings further validate that our much-evolved version of the eRapid diagnostic technology is capable of a fast, accurate, and differentiated assessment of antibodies against viral antigens in individuals," said Ingber. "We can obtain these results at extremely low cost using extremely small samples that individuals could easily self-collect and test at home or send to central laboratories. Thus, eRapid opens the opportunity of being used as a tool for pandemic surveillance and therapeutic monitoring, not only in the present but also for future pandemic and epidemic outbreaks." Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children's Hospital, and the Hansjörg Wyss Professor of Bioinspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS).

The new findings build on a previous study that showed eRapid technology is capable of simultaneously detecting SARS-CoV-2-specific RNA and antibodies on the same electrochemical sensor chips. In their new study, the team honed further in on the N protein and virus-induced immunity. Using specifically engineered eRapid sensors and a collection of 93 clinical samples of only 1.5 microliters in volume, they were able to distinguish 54 SARS-CoV-2 positive patients from 39 negative individuals within 10 minutes, with 100% sensitivity (all positive samples identified) and 100% specificity (all negative samples identified).

New diagnostic possibilities

"The combined features of eRapid make it an extremely useful platform for the fast and multiplexed detection of antibodies emerging in patients against a growing and fluctuating number of viral and other antigens, and for following an individual's antibody levels over time as we showed in our new study," said Jolly. "We took the Wyss' eRapid platform through an extensive de-risking program by engineering new nanochemistry, manufacturing, and sensing abilities. At this point, we'd like to see our technology benefit as many patients in as many disease areas as possible, including, of course, infectious diseases such as COVID-19."

In 2022, the eRapid technology was licensed to the Wyss' startup StataDX for the fields of neurological, cardiovascular, and renal diseases. First author Sanjay Sharma Timilsina, Ph.D., and second author Nolan Durr, two former members of the Wyss' eRapid team who had been instrumental in advancing the novel electrochemical sensing approach as a diagnostic platform, joined StataDX. The Wyss Institute is currently exploring additional commercial opportunities to commercialize eRapid for multiple other application areas including infectious disease diagnostics.

"With strides that we are making in parallel on developing portable devices for housing eRapid diagnostic assays, we believe that eRapid could serve as one of the first multiplexed diagnostic platforms for a wide variety of diagnostic applications as it is based on electrochemical detection and so functions much like the glucometer that is already used world-wide for patients with diabetes," said Ingber.

The study was funded by the Wyss Institute for Biologically Inspired Engineering at Harvard University and GBS Inc.

Source: Wyss Institute for Biologically Inspired Engineering at Harvard

Reference:

Timilsina, S.S., et al. (2022) Rapid quantitation of SARS-CoV-2 antibodies in clinical samples with an electrochemical sensor. Biosensors and Bioelectronics. doi.org/10.1016/j.bios.2022.115037.

Saturday, January 21, 2023

Label-free SERS Technology Realizes Rapid Identification of Beer Spoilage Bacteria

In a study published in Analytical Methods, a research group led by LI Bei from the Changchun Institute of Optics, Fine Mechanics and Physics (CIOMP) of the Chinese Academy of Sciences (CAS) proposed rapid detection of beer spoilage bacteria based on label-free surface-enhanced Raman spectroscopy (SERS) technology.

Lactic acid bacteria are common spoilage bacteria in beer and need to be monitored and controlled at all stages of beer production. Traditional spoilage bacteria detection methods are time-consuming and cannot meet the demand for real-time, in-situ, rapid detection during the production process.

Raman spectroscopy has been widely used for microbial detection due to its fast, non-destructive and accurate properties, but conventional Raman spectroscopy has the disadvantage of poor signal-to-noise ratio, which affects the accuracy of microbial identification.

Compared with conventional Raman spectroscopy, the SERS technique has a stronger and more sensitive signal and is well suited to the detection of beer spoilage bacteria. Furthermore, the label-free SERS technique is ideal for commercialization due to its low cost and good results.

In this study, the researchers improved the existing process for the preparation of label-free SERS silver nanoparticles (AgNPs). The effect of the AgNPs@KCl agglomeration effect on SERS enhancement was investigated. Eight species of beer spoilage bacteria produced during the beer brewing process were identified by SERS.

The researchers further investigated the effect of the method on the final identification rate by combining the t-distributed stochastic neighbor embedding (t-SNE) dimensionality reduction analysis algorithm, Support Vector Machine (SVM), k-NearestNeighbor (KNN) and Linear Discriminant Analysis (LDA) machine learning algorithms. All three machine learning algorithms achieved an accuracy of around 90% and performed well in identifying beer spoilage bacteria.

In the stability analysis and mixing tests, two known spoilage bacteria were mixed with pure beer and incubated at constant temperature for a period of time to identify the bacteria in the beer. The two spoilage bacteria were successfully detected in the samples and had good spectral stability.

According to the final validation study, the technique can indeed identify the target spoilage bacteria from the simulated samples, which is of great significance to the rapid identification of spoilage bacteria in beer brewing process.

Reference

Lindong Shang et al, Rapid detection of beer spoilage bacteria based on label-free SERS technology, Analytical Methods (2022). DOI: 10.1039/D2AY01221A 

Wednesday, January 18, 2023

RT-PCR Could be a More Rapid Test for C. Difficile on Environmental Surfaces

There might be a new way to detect Clostridiodes difficile on surfaces rapidly, which could be especially crucial in hospital settings.

A team, led by Rachel J. Grainger, Department of Clinical Microbiology, RCSI Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, compared the detection of C. difficile by RY-PCR to culture-based strategies and determined which is more sensitive and specific in a clinical environment.

Detecting C. difficile

Detection of C. difficile from the environment is generally done using bacterial culture, which can take several days to receive results. In addition, environmental surveillance is not usually done routinely and only conducted during outbreak investigations or as part of research.

“Environmental surveillance for Clostridioides difficile is challenging,” the authors wrote. “There are no internationally agreed recommendations on which method should be used when environmental surveillance is undertaken.”

Currently, culture-based tests are the most commonly used testing methods but can take 48 hours for results. Molecular methods also offer lower TATs, but these methods are not yet optimized.

There is a need for more rapid methods to detect C. difficile, which could aid in preventing infections.

“Unlike culture for CD, which takes too long, molecular methods such as PCR have potential in monitoring the patient environment in hospitals for CD and might be used to provide reassurance to patients and hospitals before patients are admitted to a hospital bed,” the authors wrote.

The study included 44 near patient areas of patients who are C. difficile positive, each 1 sampled using contact plates and moistened flocked swabs. The investigators took environmental samples over a 6-month period in an adult 820-bed tertiary referral hospital from the floor of the room, the bed rail, the tray table, the call bell, the mattress, the toilet floor, the toilet flush handle, and the internal bathroom door handle.

Finding Results From Samples

There were 352 samples taken using flocked swabs, resulting in 59 positive samples. This was more than the 35 positive samples found with alcohol treated and sub-cultured onto C. difficile selective agar (P = 0.01).

Moreover, there were 23 samples positive using both culture methods, 36 samples positive using the CHROMID agar only, and 12 samples positive using the alcohol treatment and culture method only.

There were 71 samples culture-positive for C. difficile using the flocked swab method compared to 29 samples using the contact plates.

The results also show 5.43% (n = 19) of samples were positive using both methods, 14.86% (n = 52) were positive only by the flocked swab and culture-method, and 2.86% (n = 10) were positive only by contact plates.

Finally, 15.14% (n = 53) of samples were positive using both the tcdB RT-PCR assay and the culture-based method, while 4.86% (n = 17) were positive using the RT-PCR negative for tcdB and 7.43% (n = 26) were RT-PCR positive for tcdB but culture-negative.

The investigators also analyzed positive samples taken from specific area of the infected patient areas.

The surface with C. difficile most frequently found on it was mattresses (n = 15; 36 %), followed by room floors (n = 14; 29 %) and toilet floors (n = 9; 31 %) using a flocked swab followed by RT-PCR for tcdB.

However, after using the flocked swab sampling technique and culture, the investigators detected C. difficile from mattresses (n = 11; 26 %), toilet floors (n = 8; 28 %), and room floors (n = 13; 27 %). When they used contact plates, they detectedC. difficile most frequently from the room floor (n = 5; 10 %), toilet floor (n = 7; 24 %), and the arm of armchair (n = 4; 27 %).

The results show detection using moistened flocked swabs followed by RT-PCR or culture resulted in more C. difficile detected compared to using the contact pates. The sensitivity of a RT-PCR assay for tcdB was 76% compared to the culture methods, while the specificity was 91%.

“Despite the lower sensitivity and specificity, RT-PCR could potentially offer a more rapid and practical alternative,” the authors wrote. “While culture picked up more positive samples for CD, PCR detected about three-quarters of the positive samples but the results from PCR were available within hours and not days.”

The investigators suggested future research should focus on confirming the role of PCR in the prevention and control of C. difficile in hospitals.

The study, “A comparison of culture methods and polymerase chain reaction in detecting Clostridioides difficile from hospital surfaces,” was published online in the Journal of Medical Microbiology.

Diagnostic Aid Rapidly Identifies Respiratory Pathogens in Critically ill Children

A molecular diagnostic aid provides reliable and fast respiratory pathogen identification in mechanically ventilated children with pneumonia.

A diagnostic aid based on polymerase chain reactions (PCR), that uses a 52-pathogen custom array card, has been found to provide both rapid (compared to blood culture) and reliable information on respiratory infections in critically ill, mechanically ventilated children, according to a study by UK researchers.

Respiratory tract infections are responsible for a large number of admissions to paediatric intensive care units. Moreover, an intensive care unit is unique environment and for which clinicians often make decisions to use antibiotics with some degree of diagnostic uncertainty. This was clearly illustrated in one study of paediatric intensive care unit children, where despite most critically children receiving antimicrobial therapy, infection was often not microbiologically confirmed. While in many cases, respiratory infections are viral in nature, it is necessary to utilise methods such as quantitative PCR, as a diagnostic aid to identify the presenting pathogens. In fact, a recent study in adults found that multiplex bacterial PCR examination of bronchoalveolar lavage, reduced the duration of inappropriate antibiotic therapy of patients admitted to hospital with pneumonia and who were at risk of Gram-negative infection. 

In the current study, researchers made use of the TaqMan Array Card (TAC) as a diagnostic aid which is a microfluidic quantitative PCR system comprising of 384 wells containing pre-aliquoted customised primer and probe combinations. The aid has been previously shown to be of value in supporting ventilator-associated pneumonia (VAP) diagnosis in adults. Nevertheless, it has not been examined in critically ill children and therefore, the aim of the present study was to assess the utility of TAC to identify bacterial and fungal respiratory pathogens in critically ill children with suspected community acquired pneumonia or VAP. The study recruited children ≤ 18 years of age and if they were mechanically ventilated and had commenced or were commencing antimicrobial therapy for a lower respiratory tract infection. The researchers determined the sensitivity and specificity of TAC to detect bacterial and fungal pathogens causing lower respiratory tract infections and the time to a result provided by TAC compared to standard microbiology cultures. Secondary objectives included a description of the micro-organisms detected by TAC but not by microbiology culture as well as the impact of TAC on antimicrobial decision-making.

Diagnostic aid and outcomes

A total of 100 children with a median age of 1.2 years (58% male) were included in the study and of whom, 80 had suspected community acquired pneumonia and the remainder, hospital acquired pneumonia.

Bacteria were detected more frequently on TAC compared to microbiology cultures (57% vs 18%, p < 0.001)) and In addition, TAC also identified more fungi (17% vs 2%, p < 0.001).

For the detection of bacterial and fungal species, TAC had a sensitivity of 89.5% (95% CI 66.9 – 98.7) and a specificity of 97.9% (95% CI 97.2 – 98.5). The median time to obtain a result for the diagnostic aid was 25.8 hours compared to 110.4 hours for microbiological cultures and overall, TAC was significantly quicker for both positive and negative results (p < 0.001).

Finally, consultants reported a change of prescription in 47% of cases based upon TAC results. Antimicrobial therapy duration was reduced or stopped in 26% of children, extended in16% and the spectrum of treatment was broadened in 17% of cases and reduced in 17%.

The authors concluded that as a diagnostic aid, TAC can be used to reliably detect pathogens quicker than routine culture in critically ill children with suspected lower respiratory tract infections and called for future studies to incorporate antimicrobial decision support and economic analysis.

Citation

Clark JA et al. The rapid detection of respiratory pathogens in critically ill children. Crit Care, 2023

Highly Accurate Test for Common Respiratory Viruses Uses DNA as ‘Bait’

A new test that ‘fishes’ for multiple respiratory viruses at once using single strands of DNA as ‘bait’, and gives highly accurate results in under an hour, has been developed by Cambridge researchers.

The test uses DNA ‘nanobait’ to detect the most common respiratory viruses – including influenza, rhinovirus, RSV and COVID-19 – at the same time. In comparison, PCR (polymerase chain reaction) tests, while highly specific and highly accurate, can only test for a single virus at a time and take several hours to return a result.

While many common respiratory viruses have similar symptoms, they require different treatments. By testing for multiple viruses at once, the researchers say their test will ensure patients get the right treatment quickly and could also reduce the unwarranted use of antibiotics.

In addition, the tests can be used in any setting, and can be easily modified to detect different bacteria and viruses, including potential new variants of SARS-CoV-2, the virus which causes COVID-19. The results are reported in the journal Nature Nanotechnology.

The winter cold, flu and RSV season has arrived in the northern hemisphere, and healthcare workers must make quick decisions about treatment when patients show up in their hospital or clinic.

“Many respiratory viruses have similar symptoms but require different treatments: we wanted to see if we could search for multiple viruses in parallel,” said Filip Bošković from Cambridge’s Cavendish Laboratory, the paper’s first author. “According to the World Health Organization, respiratory viruses are the cause of death for 20% of children who die under the age of five. If you could come up with a test that could detect multiple viruses quickly and accurately, it could make a huge difference.”

For Bošković, the research is also personal: as a young child, he was in hospital for almost a month with a high fever. Doctors could not figure out the cause of his illness until a PCR machine became available.

“Good diagnostics are the key to good treatments,” said Bošković, who is a PhD student at St John’s College, Cambridge. “People show up at hospital in need of treatment and they might be carrying multiple different viruses, but unless you can discriminate between different viruses, there is a risk patients could receive incorrect treatment.”

PCR tests are powerful, sensitive and accurate, but they require a piece of genome to be copied millions of times, which takes several hours.

The Cambridge researchers wanted to develop a test that uses RNA to detect viruses directly, without the need to copy the genome, but with high enough sensitivity to be useful in a healthcare setting.

“For patients, we know that rapid diagnosis improves their outcome, so being able to detect the infectious agent quickly could save their life,” said co-author Professor Stephen Baker, from the Cambridge Institute of Therapeutic Immunology and Infectious Disease. “For healthcare workers, such a test could be used anywhere, in the UK or in any low- or middle-income setting, which helps ensure patients get the correct treatment quickly and reduce the use of unwarranted antibiotics.”

The researchers based their test on structures built from double strands of DNA with overhanging single strands. These single strands are the ‘bait’: they are programmed to ‘fish’ for specific regions in the RNA of target viruses. The nanobaits are then passed through very tiny holes called nanopores. Nanopore sensing is like a ticker tape reader that transforms molecular structures into digital information in milliseconds. The structure of each nanobait reveals the target virus or its variant.

The researchers showed that the test can easily be reprogrammed to discriminate between viral variants, including variants of the virus that causes COVID-19. The approach enables near 100% specificity due to the precision of the programmable nanobait structures.

“This work elegantly uses new technology to solve multiple current limitations in one go,” said Baker. “One of the things we struggle with most is the rapid and accurate identification of the organisms causing the infection. This technology is a potential game-changer; a rapid, low-cost diagnostic platform that is simple and can be used anywhere on any sample.”

A patent on the technology has been filed by Cambridge Enterprise, the University’s commercialisation arm, and co-author Professor Ulrich Keyser has co-founded a company, Cambridge Nucleomics, focused on RNA detection with single-molecule precision.

“Nanobait is based on DNA nanotechnology and will allow for many more exciting applications in the future,” said Keyser, who is based at the Cavendish Laboratory. “For commercial applications and roll-out to the public we will have to convert our nanopore platform into a hand-held device.”

“Bringing together researchers from medicine, physics, engineering and chemistry helped us come up with a truly meaningful solution to a difficult problem,” said Bošković, who received a 2022 PhD award from Cambridge Society for Applied Research for this work.

The research was supported in part by the European Research Council, the Winton Programme for the Physics of Sustainability, St John’s College, UK Research and Innovation (UKRI), Wellcome, and the National Institute for Health and Care Research (NIHR) Cambridge Biomedical Research Centre.

Reference:

Filip Bošković et al. ‘Simultaneous identification of viruses and viral variants with programmable DNA nanobait.’ Nature Nanotechnology (2022). DOI: 10.1038/s41565-022-01287-x

Friday, December 30, 2022

New Methods Can Advance the Development of Rapid Diagnostics for Many Infectious Diseases

Two companion studies report new artificial intelligence approaches for identification of molecular "fingerprints" for specific infections. The methods, used here to derive a fingerprint for COVID-19, can advance the development of rapid diagnostics for many infectious diseases.

The first manuscript provides, for the first time, a framework for systematic quantification of the robustness and cross-reactivity of a potential group of blood biomarkers that act as a signature for infectious disease diagnosis. Robustness is the ability of a signature to detect a disease state (e.g., COVID-19) consistently in multiple independent groups. Cross-reactivity is the undesired extent to which a signature incorrectly detects an unintended infection or condition (e.g., a COVID-19 signature falsely detects an influenza infection). The framework was developed through curation and integration of a massive public data collection and application of a standardized signature scoring method. Applying the framework to published and synthetic signatures, the researchers demonstrate an inherent trade-off between robustness and cross-reactivity.

The second manuscript addresses the limitations of previous signature discovery approaches by modeling the robustness/cross-reactivity tradeoff with multi-objective optimization. The researchers apply this new method to identify a highly-specific blood-based signature for SARS-CoV-2 infection, which they validated in multiple independent cohorts. Interpretable signatures are more likely to be robust because they capture a reproducible biological process, such as antiviral response or cytokine signaling. Consonant with this insight, they show that the COVID-19 signature is interpretable as a combination of signals from two kinds of immune cells, plasmablasts (a rapidly proliferating, antibody-producing cell) and memory T cells (antigen-specific T cells that remain long after an infection has been eliminated). In analysis of single cell gene expression data, they found that plasmablasts mediate COVID-19 detection and memory T cells control against cross-reactivity of the signatures with other viral infections.

Standard tests for infection diagnosis involve a variety of technologies such as microbial cultures and PCR assays. These standard tests share a common design principle, which is to directly quantify pathogen material in patient samples. As a consequence, standard tests can have poor detection, particularly early after infection, before the pathogen replicates to detectable levels. For example, due to insufficient viral genetic material, PCR-based tests for SARS-CoV-2 may miss more than 60 percent of cases within the first few days of infection. To overcome these limitations, new tools for infection diagnosis are urgently needed.

The study was conducted by researchers at the Icahn School of Medicine at Mount Sinai, and Yale School of Medicine, and is published December 21 in Cell Systems.

Upon infection, an individual's immune system rapidly responds. This response includes the transcription of many genes coding for proteins that help combat the pathogen. The activated group of genes serves as a 'fingerprint' for the particular infection and is referred to as a host response signature. Host response signatures to infection have emerged as an intense area of research due to their potential both to improve understanding of pathogenesis and to provide a new paradigm for diagnosis.

The performance and overall clinical applicability of host response signatures depend on two main properties, robustness and cross-reactivity. Robustness is the ability of a signature to detect a disease state (e.g., COVID-19) consistently in multiple independent cohorts. Cross-reactivity is the undesired extent to which a signature incorrectly detects an unintended infection or condition (e.g., a COVID-19 signature falsely detects an influenza infection). Achieving the full potential of host response signatures requires both gains in robustness and reduction in cross-reactivity.

The two manuscripts present fundamental advances in the field. The first study curates massive public data and establishes a framework for systematic candidate signature evaluation. The second study leverages this framework and develops a multi-objective optimization approach to identify a highly robust and not cross-reactive COVID-19 signature.

This work was supported by the Defense Advanced Research Projects Agency (DARPA) through the Epigenetic Characterization and Observation (ECHO) program (Contract nr. XYZ) and the Defense Health Agency through the Naval Medical Research Center.

Publication Links:

Benchmarking transcriptional host response signatures for infection diagnosis

Multi-objective optimization identifies a specific and interpretable COVID-19 host response signature

Source: News Medical Life Sciences

Wednesday, December 14, 2022

New Liquid-Coated Air Filters can Improve Early Detection and Analysis of Airborne Pathogens

Researchers from the University of Maine and University of Massachusetts Amherst have designed new liquid-coated air filters that allow for improved early detection and analysis of airborne bacteria and viruses, including the one that causes COVID-19. 

While conventional air filters help control the spread of disease in public spaces like hospitals and travel hubs, they struggle to keep the pathogens they capture viable for testing. The inefficiency can inhibit scientists’ ability to identify biological threats  early on, which could hinder any response and protection measures. 

The research team, led by Caitlin Howell, a UMaine associate professor of biomedical engineering, developed a composite membrane with a liquid layer for filters that is better suited for capturing viable bacterial and viral samples for analysis. They modeled the membrane after the Nepenthes pitcher plant, which has a slippery rim and inner walls that cause insects to fall and become trapped within its digestive fluid. By keeping the bacteria and viruses they capture feasible for examination, researchers say their novel liquid-coated air filters can enhance air sampling efforts, early pathogen detection and biosurveillance for national security.    

“I think for our patients and ourselves as caregivers, this technology will give us the confidence we are safer in performing care,” says Dr. Robert Bowie, medical director of the Down East Emergency Medical Institute. “Knowing we have improved safety makes it easier to leave our loved ones and go to work caring for others.”

The group of researchers developed multiple types of filters that contained their liquid-coated membrane technology, and tested their ability to preserve and release E. coli bacteria; SARS-COV-2, the virus that causes COVID-19; and JC polyomavirus, which attacks the central nervous system. 

They specifically found that more airborne pathogens were captured by high efficiency particulate air (HEPA) filters with their liquid-coated membrane than those without. The team published their findings in the journal ACS Applied Materials & Interfaces, under the publication title, "Improved Recovery of Captured Airborne Bacteria and Viruses with Liquid-Coated Air Filters." 

“During the early stages of the pandemic we were watching in real time how many problems were being caused by no one knowing where the airborne virus was and where it wasn’t. We had a system that could start to address that need, so it was our responsibility to step up and help out,” Howell says. 

The project was a significant interdisciplinary effort across the fields of biomedical engineering, chemical engineering and microbiology. The UMaine biomedical engineering team included first author and Susan J. Hunter Presidential Award winner Daniel Regan, Graduate School of Biomedical Science, Engineering (GSBSE) Ph.D. student Chun Ki Fong and former master’s student Justin Hardcastle. The microbiology team, led by associate professor Melissa Maginnis, included Avery Bond, a Ph.D. student in molecular and biomedical sciences, and Claudia Desjardins, then a university laboratory assistant in wastewater analysis. The chemical engineering team, based at UMass Amherst, consisted of professor Jessica Schiffman and Ph.D. student Shao-Hsiang Hung. The team was joined by Andrew Holmes, a biocontainment research scientist with University of Maine Cooperative Extension. 

Regan first pitched the initial concept for liquid-coated air filters to capture bacteria-containing aerosols to his dissertation committee in March of 2019, based on conversations with military researchers and concerns for detecting potential contamination during medical evacuations. He also featured it in a presentation for the 2020 UMaine Student Symposium titled “Optimizing Liquid-Gated Membranes for Bioaerosol Capture and Release, which earned him the Dr. Susan J. Hunter Presidential Research Impact Award. 

The concept was further developed and refined when Howell, Maginnis, Schiffman, and Holmes realized that this could also apply to virus-containing aerosols in the early days of the COVID-19 pandemic and applied for funding from the National Science Foundation. In 2020, the project was awarded a $225,000 NSF EAGER award — an early concept grant that supports  “untested, but potentially transformative research ideas or approaches.”

“COVID-19 has been a constant reminder of the important role biosurveillance capabilities provide for decision makers to have detailed information for reducing biological risks” says Regan, now a fellow at the Janne E. Nolan Center on Strategic Weapons, an institute of the Council on Strategic Risks in Washington, D.C. “In the last year alone, the world has experienced high-consequence pathogens including an outbreak of monkeypox (or mpox), a resurgence of Ebola Sudan and high case numbers of Respiratory Syncytial Virus Infection (RSV). The need for pathogen early warning could not be greater, and it is our hope that further investment in liquid-coated air filters can help advance biosurveillance capabilities for aerosol detection.” 

Source: University of Maine 

Accelerating Pathogen Identification in Infants and Children with Bloodstream Infections

A collaborative team led by researchers from Great Ormond Street Institute of Child Health (GOSH), London and including researchers from the Wyss Institute for Biologically Inspired Engineering at Harvard University and BOA Biomedical in Cambridge has re-engineered the process of microbial pathogen identification in blood samples from pediatric sepsis patients using the Wyss Institute’s FcMBL broad-spectrum pathogen capture technology. The advance enables accurate pathogen detection with a combination of unprecedented sensitivity and speed, and could significantly improve clinical outcomes for pediatric and older patients with bloodstream infections (BSIs) and sepsis. The findings were published in PLoS ONE with the following publication title: FcMBL magnetic bead-based MALDI-TOF MS rapidly identifies paediatric blood stream infections from positive blood cultures.

BSIs with various microbial pathogens can rapidly escalate to life-threatening sepsis when the body is overwhelmed by the multiplying invaders and shuts down its organs’ functions. In 2017, there were 48.9 million cases and 11 million sepsis-related deaths worldwide. Importantly, almost half of all global sepsis cases occurred among children, with an estimated 20 million cases and 2.9 million global deaths in those under five years of age.

To prevent BSIs from progressing to full-blown sepsis, the infection-causing bacterial or fungal species must be identified as fast as possible. Only then can optimal pathogen-tailored antibacterial or antifungal treatments be applied in time. The conventional method used in clinical laboratories to identify the causative pathogenic species is long and laborious, requiring two time-consuming culture steps that take at least 1 to 3 days to complete.

“For all patients with sepsis, their chances of surviving dramatically shrink the longer it takes to identify the infection-causing pathogen(s) and thus, receive the most promising antimicrobial treatment,” said Nigel Klein, M.D., Ph.D., pls, a Professor of Infectious Disease and Immunology at GOSH, and a senior author on the study. “At Great Ormond Street Hospital we have been working to demonstrate both the importance rapid diagnosis and the fact that with innovative approaches we can identify the causative organism in between 40 minutes and 6 hours. Compared to adult patients, sepsis in infants and small children progresses much faster, and therefore there is a real need for diagnostic methods that support early detection.  Accurate diagnosis is even more significant due to the availability of only small blood volumes from pediatric patients which can make re-sampling challenging.”

In 2020, senior authors Klein and Elaine Cloutman-Green, Ph.D., a Consultant Clinical Scientist and Infection Control Doctor at GOSH, began collaborating with Lead Staff Scientist Michael Super, Ph.D. and  Founding Director Donald Ingber, M.D., Ph.D. at Harvard’s Wyss Institute to solve this problem. “Based on our earlier success with FcMBL in isolating pathogens from joints as well as bovine and human blood with extraordinary efficiencies, we hypothesized that building an FcMBL-mediated pathogen capture into a modified clinical blood culture protocol could shorten the time and reduce the size of the required patient samples to yield the same results that time-consuming blood culture protocols provide,” said Super.

In the pathogen identification process currently performed in clinical settings, first, blood samples are added to bottles containing liquid media in which infectious microbes, if present, are amplified to a certain density. Then, the amplified microbes are grown on solid media as isolated colonies whose constituent cells eventually can be identified with a highly sensitive, yet fast and relatively inexpensive analytical method know as MALDI-TOF mass spectrometry (MS). “Indeed, isolating the infectious microbes directly from grown liquid blood cultures using FcMBL makes them available for MALDI-TOF MS analysis much earlier,” added Super.

Accelerating pathogen identification in infants and children with bloodstream infections

The genetically engineered FcMBL protein, coupled to magnetic beads, can capture more than 100 different microbial species with high efficiency, including all of the bacterial and fungal pathogens causing sepsis. This image shows the fungal pathogen Candida albicans (purple) isolated with the FcMBL broad-spectrum pathogen capture technology. Credit: E. Super / Wyss Institute at Harvard University

FcMBL is the key component of a broad-spectrum pathogen capture technology. It consists of a genetically engineered human immune protein called mannose-binding lectin (MBL) that is fused to the Fc fragment of an antibody molecule to produce the resulting FcMBL protein. In this configuration, the MBL portion of FcMBL can capture more than 100 different microbial species with high efficiency, including virtually all of the bacterial and fungal pathogens causing sepsis. FcMBL’s Fc portion can be used to couple it to magnetic beads, allowing the captured pathogens to be quickly pulled out of patient samples and liquid blood cultures.

In the earlier stages of the project, the Wyss team provided purified bead-coupled FcMBL to the GOSH team, which had access to blood samples from pediatric patients at the hospital. At later stages, the sepsis and infectious disease company BOA Biomedical, co-founded by Super and Ingber to commercialize the Wyss Institute’s FcMBL technology, provided the FcMBL reagent and critical expertise to the project. BOA Biomedical meanwhile developed the manufacturing capabilities for FcMBL that the Food and Drug Administration (FDA) in the US and other federal health agencies require for producing therapeutic and diagnostic products.

“Sepsis is the leading killer in hospitals, and rapidly initiating the right antibiotic saves lives. Using work originally developed at the Wyss Institute, BOA Biomedical’s revolutionary FcMBL technology helps to quickly and accurately identify the pathogen causing sepsis, ushering in a new era of targeted antimicrobial therapy to help individual patients and curb society’s deadly antimicrobial resistance problem,” stated Mike McCurdy, M.D., Chief Medical Officer of BOA Biomedical.

In addition to using the gold standard two-step blood culture in combination with MALDI-TOF MS pathogen identification, the team also included the Bruker Corporation’s MBT Sepsityper® kit as a comparison. Brought to market in 2021, the MBT Sepsityper® essentially eliminates the time-consuming second microbial culture step by lysing microbial cells from the liquid culture and spinning the fragments down in a centrifuge before analyzing them by MALDI-TOF mass spectrometry analysis. Although it accelerates the overall diagnostic process, the MBT Sepsityper® method produces lower microbial detection rates than those obtained with the conventional culture method, which means that it may still fail to identify the infection-causing pathogen in a significant fraction of blood samples.

“Our FcMBL approach has opened up the opportunity to identify pathogenic organisms to guide treatment 24 to 48 hours earlier than would be possible using standard culture techniques. It has also enabled us to use this identification to make any ongoing culture for antibiotic sensitivities more tailored to the needs of the patient. This method isn’t tied into a specific platform or manufacturer, and thus we see clear potential for it to become a new standard processing step for clinical pathogen detection,” said Cloutman-Green.

“The FcMBL method identified 94.1% of microbial species found in clinical blood culture analysis with samples from 68 pediatric patients,” said first author Kerry Kite, who performed her graduate work with Klein and Cloutman-Green. “We were able to identify more infectious species in positive liquid blood cultures using the FcMBL method than with the MBT Sepsityper® method (25 of 25 vs 17 of 25), and this trend was even further pronounced in the case of the common fungal pathogen Candida (24 of 24 vs 9 of 24).” Candida species account for about 5% of all cases of severe sepsis and are the fourth most common pathogen isolated from patients’ bloodstreams in the United States. Not only do infections with Candida and other fungi require specific antifungal treatments, distinguishing among the various types of pathogenic fungi helps direct the appropriate antimicrobial therapy. Specifically in neonatal intensive care units, Candida infections are a major cause of morbidity and mortality, killing as many as 40% of infants and often causing neurodevelopmental impairments in those that survive.

“By continuously adapting the powerful FcMBL pathogen capture technology to unmet and pressing diagnostic needs, such as the rapid diagnosis of sepsis in pediatric patients, we hope to profoundly alter the frequently dismal prospects of patients of all ages,” said Ingber. “Our ultimate goal is to be able to accurately and even more rapidly identify pathogens directly in small samples of blood without the need for any additional microbial cultures.” Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Bioinspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

The study was also authored by Sahil Loomba and Thomas Elliott at Imperial College London; Francis Yongblah, Lily Gates and Dagmar Alber at GOSH; George Downey and James Hill at BOA Biomedical; and Shanda Lightbown and Thomas Doyle at the Wyss Institute. The authors were supported in their work by the clinical microbiology staff at GOSH, as well as Erika Tranfield with MALDI-TOF MS expertise. At GOSH, critical financial help for the project from the Benecare Foundation, philanthropists Luca Albertini and Professor Pauline Barrieu, as well the Office of Vice-President (Advancement) at University College London was coordinated by Simona Santojanni. At the Wyss Institute, the study was funded by the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement Number W911NF-16-C-0050, and the Wyss Institute’s technology translation engine. Additional support was provided by BOA Biomedical.

Source: Wyss Institute

Birmingham Researcher Wins WH Pierce Award for Work on Rapid, Portable Methods for the Detection of Antibiotic Resistance

Organised by Applied Microbiology International, the awards celebrate the brightest minds in the field and promote the research, groups, projects, products and individuals who are shaping the future of applied microbiology.

The guest of honour was Professor Sir Jonathan Van-Tam MBE, Pro-Vice-Chancellor for the Faculty of Medicine & Health Sciences at the University of Nottingham who was named an Honorary Fellow at the prestigious event.

The WH Pierce Prize was founded in 1984 in memory of the late WH (Bill) Pierce, former Chief Bacteriologist of Oxo Ltd and a long-time member of the Society. It is awarded to a scientist who has used microbiology to make a significant contribution to One Health advancements.

The prize was awarded to Joshua Quick of the University of Birmingham, whose project OneAMR aims to develop rapid, portable methods for the detection of antibiotic resistance by combining single-cell genomics and nanopore sequencing technology.

Joshua launched his career in industry working on the development of Illumina DNA sequencing platforms including the MiSeq. After meeting Nick Loman he moved to Birmingham to start a PhD in Biological Sciences in 2012.

During his PhD, Joshua applied whole-genome sequencing to investigate hospital outbreaks of Pseudomonas aeruginosa and Salmonella enterica. He travelled to Guinea during the West African Ebola outbreak to establish the first real-time, genomic surveillance laboratory. He has also travelled to Brazil, DRC and São Tomé with a lab-in-a-suitcase for research or WHO deployments.

Joshua developed an ultra-long fragment method for sequencing reads of up to 1 million basepairs using nanopore. This was used to assemble the E. coli genome in 8 reads and later to generate the first telomere-to-telomere human genome assembly.

He developed a method for viral genome sequencing which includes a website to design primers and laboratory protocols to generate genome sequences. The primalscheme website has become a vital community resource, processing 30,000 jobs a year. The lab protocol is number 1 out of 13,000 protocols on protocols.io, the protocols sharing website.

In total, more than 10 million genomes have been generated using the method which has been commercialised by multiple companies including Illumina, NEB and Qiagen.

In 2019 Joshua was awarded a 7-year UKRI FLF Fellowship to establish his own lab. His project, OneAMR aims to develop rapid, portable methods for the detection of antibiotic resistance, a serious global threat, by combining single-cell genomics and nanopore sequencing technology.

Source: University of Birmingham