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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Impedance Cytometry for Rapid Antibiotic Susceptibility Testing

Scientists at Nara Institute of Science and Technology in Japan have come up with a method to rapidly determine the antibiotic susceptibility of a bacterial sample, such as a patient sample from a non-healing infected wound. The technique is based on impedance cytometry, which involves a high-throughput single cell analysis of the bacterial cells. The impedance system measures the dielectric properties of the cells as they flow through the device, and it can assess up to 1000 cells per minute.

Using machine learning to determine the differences in the dielectric properties between samples that have been treated with antibiotics and untreated samples let the researchers identify the susceptibility of the bacteria to a given antibiotic in as little as two hours after treatment.

Antibiotic-resistant bacteria are becoming more common, and the consequences for our healthcare will be profound. Routine surgical procedures could become fraught with risk and simple infections could progress into more serious issues without any available drugs to combat them. However, thankfully, we are not at this stage quite yet, and we still have time to prolong the utility of our existing antibiotic arsenal. In this context, using the correct antibiotic drug is important to achieve the desired treatment outcome, and also to reduce the likelihood that resistance develops further  

Getting a readout on the antibiotic susceptibility of the bacteria causing problems for a particular patient is best completed quickly so that the patient can avail of the correct treatment as soon as possible. However, current approaches to achieve this can take too long. “Oftentimes susceptibility results are needed much faster than conventional tests can deliver them,” said Yaxiaer Yalikun, a researcher involved in the study. “To address this, we developed a technology that can meet this need.”

The new technology involves using impedance cytometry to measure the dielectric properties of the bacteria. These properties will change quite quickly on contact with an antibiotic that the bacteria are susceptible to. The researchers split the bacterial sample in two, and treat one of these samples with an antibiotic before separately analyzing treated bacteria and untreated controls. Then, a machine learning algorithm learns the characteristics of the untreated bacteria, and determines if there is anything different with the treated cells.  

“Although there was a misidentification error of less than 10% in our work, there was a clear discrimination between susceptible and resistant cells within 2 hours of antibiotic treatment,” said Yoichiroh Hosokawa, another researcher involved in the study.

Reference:

Tao Tang, Xun Liu, Yapeng Yuan, Ryota Kiya, Tianlong Zhang, Yang Yang, Shiro Suetsugu, Yoichi Yamazaki, Nobutoshi Ota, Koki Yamamoto, Hironari Kamikubo, Yo Tanaka, Ming Li, Yoichiroh Hosokawa, Yaxiaer Yalikun. Machine learning-based impedance system for real-time recognition of antibiotic-susceptible bacteria with parallel cytometry. Sensors and Actuators B: Chemical. Volume 374. 2023.

Abstract: 

Impedance cytometry has enabled label-free and fast antibiotic susceptibility testing of bacterial single cells. Here, a machine learning-based impedance system is provided to score the phenotypic response of bacterial single cells to antibiotic treatment, with a high throughput of more than one thousand cells per min. In contrast to other impedance systems, an online training method on reference particles is provided, as the parallel impedance cytometry can distinguish reference particles from target particles, and label reference and target particles as the training and test set, respectively, in real time. Experiments with polystyrene beads of two different sizes (3 and 4.5 µm) confirm the functionality and stability of the system. Additionally, antibiotic-treated Escherichia coli cells are measured every two hours during the six-hour drug treatment. All results successfully show the capability of real-time characterizing the change in dielectric properties of individual cells, recognizing single susceptible cells, as well as analyzing the proportion of susceptible cells within heterogeneous populations in real time. As the intelligent impedance system can perform all impedance-based characterization and recognition of particles in real time, it can free operators from the post-processing and data interpretation.

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A Simple Label-Free Method Reveals Bacterial Growth Dynamics and Antibiotic Action in Real-Time

Scientists have published a paper in Nature that describes a simple label-free method that reveals bacterial growth dynamics and antibiotic action in real-time. They discuss a patented technology which utilizes an innovative combination of laser light scattering, locked signal and integrating detection space. The methodology, named scattered light integrated collection (SLIC), provides a very sensitive way to detect microorganisms at low concentrations allowing us to follow their growth in real-time and to study the impact of different stresses on their growth dynamics. The paper may be accessed by clicking here.

Reference

Hammond, R.J.H., Falconer, K., Powell, T. et al. A simple label-free method reveals bacterial growth dynamics and antibiotic action in real-time. Sci Rep 12, 19393 (2022). https://doi.org/10.1038/s41598-022-22671-6. 

Abstract

Understanding the response of bacteria to environmental stress is hampered by the relative insensitivity of methods to detect growth. This means studies of antibiotic resistance and other physiological methods often take 24 h or longer. We developed and tested a scattered light and detection system (SLIC) to address this challenge, establishing the limit of detection, and time to positive detection of the growth of small inocula. We compared the light-scattering of bacteria grown in varying high and low nutrient liquid medium and the growth dynamics of two closely related organisms. Scattering data was modelled using Gompertz and Broken Stick equations. Bacteria were also exposed meropenem, gentamicin and cefoxitin at a range of concentrations and light scattering of the liquid culture was captured in real-time. We established the limit of detection for SLIC to be between 10 and 100 cfu mL−1 in a volume of 1–2 mL. Quantitative measurement of the different nutrient effects on bacteria were obtained in less than four hours and it was possible to distinguish differences in the growth dynamics of Klebsiella pneumoniae 1705 possessing the BlaKPC betalactamase vs. strain 1706 very rapidly. There was a dose dependent difference in the speed of action of each antibiotic tested at supra-MIC concentrations. The lethal effect of gentamicin and lytic effect of meropenem, and slow bactericidal effect of cefoxitin were demonstrated in real time. Significantly, strains that were sensitive to antibiotics could be identified in seconds. This research demonstrates the critical importance of improving the sensitivity of bacterial detection. This results in more rapid assessment of susceptibility and the ability to capture a wealth of data on the growth dynamics of bacteria. The rapid rate at which killing occurs at supra-MIC concentrations, an important finding that needs to be incorporated into pharmacokinetic and pharmacodynamic models. Importantly, enhanced sensitivity of bacterial detection opens the possibility of susceptibility results being reportable clinically in a few minutes, as we have demonstrated.

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Rapid Diagnostic Testing Highly Accurate for Ebola Virus Disease

Two rapid diagnostic testing methods were found to have high sensitivity and specificity for diagnosing Ebola virus disease (EVD), according to study findings published in Clinical Microbiology and Infection.

Researchers conducted a systematic review of the literature to analyze the diagnostic accuracy of rapid diagnostic tests for EVD. Publication databases were searched from inception through May 2021, and the review included only diagnostic accuracy studies conducted among a live patient population with confirmed or suspected EVD.

Among 1054 studies identified, 15 were included in the review. Of the included studies, 10 assessed lateral flow-based testing and 5 assessed polymerase chain reaction (PCR)-based testing for the rapid diagnosis of EVD. Reverse transcription (RT)-PCR testing was used as the reference test in all included studies, with variations in regard to the specific assay used.

Lateral flow-based rapid testing demonstrated and overall estimated sensitivity of 86.0% (95% CI, 86.0%-86.2%) and a specificity of 97% (95% CI, 96.1%-97.9%) for diagnosing EVD. The lowest reported sensitivity associated with lateral flow testing was 62% (95% CI, 53%-73%). The researchers found significant variation in the specificity (range, 73%-100%) of lateral flow tests used across all studies. There were 2 studies that reported a sensitivity of 100% for lateral flow testing, both of which used whole blood samples obtained from patients that were tested either at the point of care or in a laboratory.

Compared with RT-PCR testing, rapid PCR testing was highly accurate for diagnosing EVD, with a sensitivity of 96.2% (95% CI, 92.4%-98.1%) and a specificity of 96.8% (95% CI, 95.3%-97.9%).

Rapid diagnostic tests were found to be highly accurate for diagnosing EVD across a range of specimen types, including whole blood, plasma, and buccal swabs.

Limitations were the inclusion of some studies with high selection bias, the lack of data on cycle threshold counts, and potential specimen degradation among studies that evaluated specimens several years following collection.

According to the researchers, “Our findings support the use of RDTs [rapid diagnostic tests] as a ‘rule in’ test to expedite treatment and vaccination” in patients with suspected EVD.

Reference

Dagens AB, Rojek A, Sigfrid L, Plüddemann A. The diagnostic accuracy of rapid diagnostic tests for Ebola virus disease: a systematic review. Clin Microbiol Infect. Published online September 23. doi:10.1016/j.cmi.2022.09.014

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Compact Rapid Detection Kit to Test for Monkeypox Virus Shows Promising Results

In a recent report in the journal Travel Medicine and Infectious Disease, researchers introduced a compact rapid detection kit that tests for monkeypox virus using a combination of Recombinase Polymerase Amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology.

Background

The monkeypox outbreak has spread widely outside the endemic west and central African countries during 2022, and the World Health Organization (WHO) has now declared it a Public Health Emergency of International Concern. The etiological agent of monkeypox is the monkeypox virus belonging to the Orthopoxvirus genus.

The currently used test for detecting monkeypox is the polymerase chain reaction test (PCR), which amplifies Orthopoxvirus or specific monkeypox virus deoxyribonucleic acid (DNA). However, PCR is a tedious process requiring a thermocycler, trained personnel, and a laboratory.

The early detection and containment of infected individuals are imperative for controlling a disease outbreak. Immunoassays such as lateral flow strips are simpler and faster than PCRs but have higher cross-reactivity for orthopoxviruses, making the precise detection of monkeypox difficult. Therefore, a technology that rapidly detects monkeypox and provides accurate and reliable results is essential.

Detection mechanism

In the present study, the researchers developed a rapid detection kit or pocket lab to test for the monkeypox virus. The detection kit uses the principles of RPA and CRISPR technology to increase the specificity and sensitivity of the analysis. The RPA amplifies target genes of the monkeypox virus, which are scanned by the CRISPR guide ribonucleic acid (RNA) and the CRISPR-associated protein 12a (Cas12a) enzyme.

The CRISPR/Cas12sa-mediated cleavage of the sequence occurs only when the target monkeypox amplicons are detected, eliminating the possibility of non-specific signals. The cleavage results in fluorescence, indicating monkeypox-positive samples.

Pocket lab

The kit weighed 500 g, and the case was water-resistance and compact enough to fit inside a pocket. The components comprised two three-dimensional printed heating blocks — block A, which heats the samples to 80 °C for viral shell lysis, and block B, which heats the samples to 40 °C for DNA amplification.

The kit contained tubes with trehalose-protected lyophilized enzymes and sampling tubes with sodium chloride and magnesium acetate solution. A rehydration buffer containing primers and the fluorescent reporter, and an ultraviolet flashlight (UV) to detect the fluorescence were also included in the kit.

The testing process involves adding the sample to the sampling tube and using a heating block A to lyse the viral shell. The heated sample is added to the tube containing lyophilized enzymes, to which the rehydration buffer is added. Heating block B is then employed to amplify the target region. Finally, the UV flashlight is used to detect the fluorescence.

During the trial test, the researchers ran using pseudotyped monkeypox virus samples, the total time taken was 25 minutes, and samples with viral particles as low as ten were detected with accuracy.

Conclusions

To summarize, the researchers introduced a novel rapid detection kit that was compact and could be used to detect the monkeypox virus without the use of complicated instruments, trained technicians, or a laboratory set-up.

The detection kit uses the principles of RPA and CRISPR to amplify target regions of the monkeypox DNA and cleave it using the CRISPR/Cas12a complex, with a fluorescent reporter signaling the detection of monkeypox viral DNA.

The trials with the pseudotyped virus indicated successful and rapid detection of the monkeypox virus, suggesting the kit’s potential use for rapid testing of travelers and in areas that lack appropriate medical infrastructure and trained staff.

Journal reference:

Wei, J., Meng, X., Li, J., & Pang, B. (2022). Pocket lab for the rapid detection of monkeypox virus. Travel Medicine and Infectious Disease. 

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A Rapid Test that Detects Group B Streptococcus is in Development

Scientists at the University of Bath are looking for volunteers to help them develop a new test that could prevent dangerous bacterial infections in newborn babies which currently kills one baby every week in the UK.

Between 20-40 per cent of women carry a bacterium called Group B Streptococcus (GBS) in their vaginas.

This normally does not cause harm, but during pregnancy, it can be passed onto the baby, which sometimes causes life-threatening infections and can result in life-long disabilities or death.

GBS is currently the leading cause of neonatal infection – in the UK, it kills around one newborn baby a week and leaves another permanently disabled.

Professor Toby Jenkins, from the University of Bath’s Department of Chemistry, is leading a team to develop a new rapid detection system that can test mothers for GBS during labour, identifying those at risk of infection so they can be treated with antibiotics and therefore reduce the risk of the baby becoming infected.

He said: “The current test is done at 36-38 weeks of pregnancy, but this may not reflect the situation during labour, resulting in either risk to the baby, or unnecessary antibiotic use by the mother.

“Our test aims to be much faster – with results in under 45 minutes – and can be done during established labour so the result is more accurate.

“If the mother is found to have GBS at this stage, antibiotics can be immediately offered, which is an effective way of preventing transmission.”

The test consists of a vaginal swab taken from the mother which is placed in a liquid that can detect GBS.

Professor Jenkins added: “We are now looking for participants to donate two vaginal swabs so we can determine the efficacy, sensitivity and specificity of the test to GBS. Participants are completely anonymous, but would be hugely helpful to our research project.”

The team is looking for individuals born biologically female, who are over 18 years old, currently not pregnant and able to take their own samples to send anonymously to the lab.

Participation is entirely confidential: the team will not know the identity of donors.

The study has full ethics approval from REACH, is supported by the charity Group B Strep Support, and funded by the European Union Horizon 2020 project STIMULUS.

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Nanotech Bubbles Burst When They Detect Viruses in the Air

Scientists have shown that they can detect SARS-CoV-2, the virus that causes COVID-19, in the air by using a nanotechnology-packed bubble that spills its chemical contents like a broken piñata when encountering the virus.

Such a detector could be positioned on a wall or ceiling, or in an air duct, where there’s constant air movement, to alert occupants immediately when even a trace level of the virus is present.

The heart of the nanotechnology is a micelle, a molecular structure composed of oils, fats and sometimes water with inner space that can be filled with air or another substance. Micelles are often used to deliver anticancer drugs in the body and are a staple in soaps and detergents. Almost everyone has encountered a micelle in the form of soap bubbles.

A team of scientists at the Department of Energy’s Pacific Northwest National Laboratory created a new kind of micelle, one that is stamped on the surface with copies of an imprinted particle for SARS-CoV-2.

The team filled micelles with a salt capable of creating an electronic signal but that is quiescent when packed inside a micelle. When a viral particle interacts with one of the imprinted receptors on the surface, the micelle pops open, spilling the salt and sending out an electronic signal instantly.

The system acts like a signal magnifier, translating the presence of one viral particle into 10 billion molecules that together create a detectable signal. The developers say that the detector has advantages over today’s technologies; it produces a signal faster, requires a much lower level of viral particles, or produces fewer errors.

“There is a need for this kind of low-cost detection system,” said PNNL scientist Lance Hubbard, a nanotechnology specialist and an author of the paper. “Perhaps it could be implemented in schools, or in hospitals or emergency rooms before patients have been fully assessed—anywhere you need to know immediately that the virus is present.”

PNNL’s micelle technology is the product of an arduous chain of 279 separate chemical steps developed by first author Samuel Morrison together with Hubbard and other PNNL scientists.

Detecting One Viral Particle Out of Billions

The team estimates that the technology can pluck one viral particle out of billions of other particles. The detector is so sensitive that the team had a challenging time identifying the lower limit. The team used both inactivated SARS-CoV-2 viral particles and the virus’s spike protein in its tests.

While the technology detects the virus within a millisecond, the device takes an additional minute to run quality-control software to confirm the signal and prevent false alarms.

Micelles can be delicate, like a soap bubble from a child’s wand. But, under certain circumstances, scientists can make hardier micelles that spill their contents at just the right time and place—for instance, these micelles that burst open when a viral particle is detected.

The PNNL micelle is bilayer, with one polymer-coated micelle inside the other, and the entire structure immersed in water. Each micelle is about 5 microns wide. On the outer surface are several imprinted particles, made of silica, about 500 nanometers wide. Each imprint is an opportunity for a COVID-causing viral particle to bind, causing the bilayer micelle to pop open.

“Combining micelles with a technology to imprint or stamp them is not something many people have done before,” said Hubbard. “Imprinting a molecule with our molecule of interest inserts a vulnerability into the micelle—which is what we want in this case.”

Morrison, a former Marine, began this line of work hoping to develop a new way to help soldiers quickly detect explosives in combat. He connected with Hubbard, an expert in nanosynthesis. They switched the focus of the project to SARS-CoV-2 when the pandemic hit. Other possible uses of the technology include detection of fentanyl and environmental toxins.

Battelle, which manages and operates PNNL for DOE, has filed for a patent on the technology. The scientists say the technology needs to be developed further, perhaps with a licensing partner, before it can be deployed broadly.

Reference

Detection of SARS-COV-2 by functionally imprinted micelles. MRS Communications, 25 October 2022

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Face Mask Detects Viral Exposure

Scientists have created a face mask that can detect common respiratory viruses, including influenza and the coronavirus, in the air in droplets or aerosols. The highly sensitive mask, presented September 19 in the journal Matter, can alert the wearers via their mobile devices within 10 minutes if targeted pathogens are present in the surrounding air.

"Previous research has shown face mask wearing can reduce the risk of spreading and contracting the disease. So, we wanted to create a mask that can detect the presence of virus in the air and alert the wearer," says Yin Fang, the study's corresponding author and a material scientist at Shanghai Tongji University.

Respiratory pathogens that cause COVID-19 and H1N1 influenza spread through small droplets and aerosols released by infected people when they talk, cough, and sneeze. These virus-containing molecules, especially tiny aerosols, can remain suspended in the air for a long time.

Fang and his colleagues tested the mask in an enclosed chamber by spraying the viral surface protein containing trace-level liquid and aerosols on the mask. The sensor responded to as little as 0.3 microliters of liquid containing viral proteins, about 70 to 560 times less than the volume of liquid produced in one sneeze and much less than the volume produced by coughing or talking, Fang says.

The team designed a small sensor with aptamers, which are a type of synthetic molecule that can identify unique proteins of pathogens like antibodies. In their proof-of-concept design, the team modified the multi-channel sensor with three types of aptamers, which can simultaneously recognize surface proteins on SARS-CoV-2, H5N1, and H1N1.

Once the aptamers bind to the target proteins in the air, the ion-gated transistor connected will amplify the signal and alert the wearers via their phones. An ion-gated transistor is a novel type of device that is highly sensitive, and thus the mask can detect even trace levels of pathogens in the air within 10 minutes.

"Our mask would work really well in spaces with poor ventilation, such as elevators or enclosed rooms, where the risk of getting infected is high," Fang says. In the future, if a new respiratory virus emerges, they can easily update the sensor's design for detecting the novel pathogens, he adds.

Next, the team hopes to shorten the detection time and further increase the sensitivity of the sensor by optimizing the design of the polymers and transistors. They are also working on wearable devices for a variety of health conditions including cancers and cardiovascular diseases.

"Currently, doctors have been relying heavily on their experiences in diagnosing and treating diseases. But with richer data collected by wearable devices, disease diagnosis and treatment can become more precise," Fang says.

The work is supported by National Key Research and Development Program, National Natural Science Foundation of China, Science and Technology Commission of Shanghai Municipality, Shanghai Municipal Science and Technology Major Project and the Fundamental Research Funds for the Central Universities.

Journal Reference

Bingfang Wang, Deqi Yang, Zhiqiang Chang, Ru Zhang, Jing Dai, Yin Fang. Wearable bioelectronic masks for wireless detection of respiratory infectious diseases by gaseous media. Matter, 2022.

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Blood-Based Diagnostic Cuts Time to Infection Detection

U.S researchers have developed a rapid processing system that can dramatically improve the time taken to detect bacterial and fungal infections in the bloodstream, potentially allowing faster administration of antibiotics.

The culture-free, “biphasic” approach enables pathogen DNA to be amplified directly from just 1 ml of whole blood, reducing the time needed for a result to less than 2.5 hours compared with more than 20 hours using conventional methods.

The system offers single-molecule sensitivity in detecting pathogens including methicillin-resistant and methicillin-sensitive Staphylococcus aureus, Escherichia coli, and Candida albicans.

Validation studies using 63 whole-blood samples showed total agreement in sensitivity and specificity with clinical laboratory results that used blood culture and polymerase chain reaction (PCR).

The new system, described in the Proceedings of the National Academy of Sciences, involves rapidly drying blood and creating a porous microfluidic and nanofluidic network within this.

DNA amplification enzymes and primers are then able to diffuse into the dried blood matrix to access any pathogen DNA within, initiating its amplification without the need for conventional nucleic acid purification.

Researcher Rashid Bashir, professor of bioengineering at the University of Illinois at Urbana-Champaign, U.S.A., told Inside Precision Medicine that traditional blood culture could take many days to provide enough bacteria for subsequent PCR detection.

“Alternatively, our approach using a new blood drying technique can be used to detect pathogens in less than a few hours. This can potentially be very important for rapid and early detection of onset of sepsis caused by bacteremia.”

Antibiotic therapy within three hours of initial, symptom-based recognition can significantly reduce the risk of death from bloodstream infections and bacteremia, the researchers note.

However, the current clinical gold standard for diagnosing sepsis and bloodstream infections remains blood culture followed by nucleic acid amplification and detection using PCR.

“The blood culture step is too slow and cumbersome to allow for initial management of patients and thus contributes to high mortality,” the authors explain.

“Moreover, in the absence of timely results from robust diagnostic tests, the patients are administered highly potent broad-spectrum antibiotics without any patient stratification, increasing antimicrobial resistance and emergence of drug-resistant and atypical pathogens.”

The new platform uses whole blood, which can be dried in as little as 10 minutes using high temperatures of 95 °C. The dried blood then acts as a substrate that does not take part in the reaction, and inhibitory elements such as platelets, cells and proteins are neutralized to form part of this substrate.

Thermal lysis improves the porosity of microfluidic and nanofluidic networks within the dried blood matrix, which allows enzymes to access pathogen DNA and initiate amplification with single-molecule sensitivity, thereby bypassing the need for conventional DNA purification.

The dried blood solid phase does not re-mix with the supernatant and keeps the high heme locked in red blood cells in the background while amplicons diffuse out and bind to fluorescent dye in the clear supernatant phase, leading the researchers to term this “biphasic amplification”.

Reporting their findings, they propose: “The reduction in instrumentation complexity and costs compared to blood culture and alternate molecular diagnostic platforms can have broad applications in healthcare systems in developed world and resource-limited settings.”

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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

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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.

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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/) 

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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.

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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

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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.

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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 

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