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

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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 

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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

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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 

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