Researchers Develop At-Home Device for Diagnosing Gingivitis and Periodontitis Caused by Bacteria

Engineers at the University of Cincinnati have developed a new device that can warn consumers about early risks of tooth decay from diseases such as gingivitis and periodontitis.

Gingivitis, the earliest form of gum disease, is caused by bacteria. But not just any bacteria.

The problem for researchers was getting a device to single out the particular type responsible for the disease, said Andrew Steckl, an Ohio Eminent Scholar and distinguished research professor in UC’s College of Engineering and Applied Science.

“It’s been quite the challenge to get to the point where we can detect this toxin created by the bacteria responsible for gingivitis,” he said.

Steckl and UC Senior Research Associate Daewoo Han collaborated with Sancai Xie, a principal scientist at Procter & Gamble Co., and described their results in a paper published in the Royal Society of Chemistry journal Sensors and Diagnostics.

Steckl’s research team has been exploring biosensing for various applications. They studied stress hormones in sweat in collaboration with the Air Force Research Lab at Wright-Patterson Air Force Base. Now they are studying saliva.

“There are good reasons to use saliva,” he said. “It’s relatively plentiful and easy to obtain through noninvasive methods. And saliva has a lot of important elements that can act as indicators of your health.”

Bacteria from gingivitis can travel through the bloodstream, leading to cardiovascular disease and other serious health problems, Steckl said.

But saliva is a complicated biofluid, Han said.

“We wanted to target a biomarker in saliva. But saliva is hard to use,” said Han, the study’s lead author.

Researchers pretreated the sample using potato starch to remove a protein called amylase that could interfere with the test results. Their test uses antibodies that react to the endotoxins found in the bacteria.

Developing a sensor required precise selectivity and sensitivity, Steckl said.

“Daewoo worked very hard on many dead-ends before he had success,” Steckl said. “I tell my students that research is search, search and re-search until you find the answer.”

At-home health testing has been available for generations for uses such as detecting pregnancy. But the COVID-19 pandemic introduced a wide audience of consumers to the concept of monitoring their health with new technology.

The at-home testing industry is expected to generate $45 billion annually by 2031, according to Allied Market Research.

Steckl said he sees a lot of opportunity for new consumer products.

“Our results definitely show promise,” Steckl said. “Sometimes it comes easy. Most of the time you have to persevere.”

Reference:

Salivary endotoxin detection using combined mono/polyclonal antibody-based sandwich-type lateral flow immunoassay device. Daewoo Han, Sancai Xieb and Andrew J. Steckl. Send. Diagn. 2023. DOI: 10.1039/d3sd00158j. 

Abstract

A point-of-care/use lateral flow assay (LFA) is reported for the detection of P. gingivalis endotoxin, a major saliva biomarker for oral health. Two different approaches of sandwich LFA design using either the combination of mono- and polyclonal antibodies or polyclonal antibody only have been evaluated to detect P. gingivalis endotoxins, having a limit of detection of ∼22 ng mL−1 and 46.5 ng mL−1 for water- and saliva-based samples, respectively. The LFA also exhibits good selectivity to P. gingivalis endotoxin versus other endotoxins and proteins. Saliva pretreatment combining syringe filtration and potato starch successfully inhibits α-amylase activity and provides improved results on LFA devices.

Read more

TU Dresden Researchers Develop Highly Innovative Solutions for the Detection of Viral Pathogens

The outbreak of the COVID pandemic in 2020 has once again shown how important reliable and rapid detection methods are to initiate effective measures to combat a pandemic. Scientists from the Chair of Materials Science and Nanotechnology at TU Dresden (TUD) have made considerable progress in the development of highly innovative solutions for the detection of viral pathogens in two studies they presented recently. The results of their work have now been published in the journals "ACS Applied Materials & Interfaces" and "Advanced Materials Interfaces".  

Customized, powerful and adaptable nanoelectronic sensors represent a promising approach to be ready to fight both current and future pandemics. These sensors not only enable conventional diagnosis in cases of suspected outbreaks, but also a continuous monitoring of ambient air in buses, trains, schools or healthcare facilities. This means that appropriate and immediate measures can be taken as soon viruses appear.

Since 2020, the Dresden scientists have been working intensively on the development of miniaturized sensors for the accurate and efficient detection of SARS-CoV-2 antigens. In addition to the TUD team led by Prof. Gianaurelio Cuniberti and Dr. Bergoi Ibarlucea, scientists from the European Molecular Biology Laboratory (EMBL) in Hamburg, the Leibniz Institute of Polymer Research (IPF) Dresden and the Pohang University of Science and Technology (POSTECH) in Korea were also involved in the two studies.

Sybodies: a revolution in biological recognition

The first study, published in the journal ACS Applied Materials & Interfaces, describes a groundbreaking innovative approach that significantly increases accuracy and speed of SARS-CoV-2 antigen detection. It involves inserting synthetic nanobodies, known as sybodies, into biosensors as receptors. "Sybodies represent a rapid, sustainable and ethically sound alternative that, unlike conventional antibodies, is developed and manufactured using non-animal methods," said Prof. Gianaurelio Cuniberti, who coordinated both studies with Dr. Bergoi Ibarlucea. "Another key advantage of using sybodies is their smaller size compared to antibodies, so biological recognition processes can take place much closer to the sensor surface, increasing signal strength and making the sensors much faster and more sensitive," he adds. Initial tests have been successfully conducted with silicon nanowire-based field-effect transistors modified with sybodies, demonstrating the great application potential of this approach.

Overcoming the loss of sensitivity in biological fluids

In another paper published in the journal Advanced Materials Interfaces, the team is looking at increasing the sensitivity of the sensors when they operate in biological fluids. Such samples have a complex molecular composition, which severely limits the sensor's detection range. To solve this problem, the scientists developed a special surface modification with a hydrogel based on the dielectric polymer polyethylene glycol. This allows measurements to be taken directly in saliva and other samples from patients, and eliminates the need for time-consuming and costly sample preparation steps.

References:

C. Zhang, A. Parichenko, W. Choi, S. Shin, L. A. Panes-Ruiz, D. Belyaev, T. F. Custódio, C. Löw, J.-S. Lee, B. Ibarlucea, G. Cuniberti, Sybodies as Novel Bioreceptors toward Field- Effect Transistor-Based Detection of SARS-CoV-2 Antigens. ACS Applied Materials and Interfaces 15, 40191 (2023). https://doi.org/10.1021/acsami.3c06073

A. Parichenko, W. Choi, S. Shin, M. Schlecht, R. Gutierrez, T. F. Akbar, C. Werner, J. Lee, B. Ibarlucea, and G. Cuniberti, Hydrogel-Gated Silicon Nanotransistors for SARS-CoV-2 Antigen Detection in Physiological Ionic Strength. Advanced Materials Interfaces (2023). https://doi.org/10.1002/admi.202300391

Read more

Japan Researchers Advance Rapid Bacterial Testing Tech to Prevent Food Poisoning

A new technology that speeds up bacterial testing in food is showing promise to “revolutionize” the process of testing bacterial viability in food, according to Japan-based scientists who discovered the breakthrough in food safety. 

They have used tetrazolium salt (MTT) to reduce the time taken to measure the number of viable bacteria in food electrochemically from two days to approximately one hour, irrespective of the bacterial species.

The technique can reportedly verify food safety before shipment from factories and prevent food poisoning – a major breakthrough, they note. 

WHO states that food safety, nutrition and food security are inextricably linked, with an estimated 600 million people falling ill and 420,000 dying each year due to contaminated food consumption. 

Food safety and hygiene in the food industry is of paramount importance.

Dr. Hiroshi Shiigi, professor and research lead at the Department of Applied Chemistry, Osaka Metropolitan University states, “In this study, we focused on the electrochemical properties of tetrazolium salts and developed a simple method for evaluating viable bacterial counts as an indicator of hygiene control in food and pharmaceutical production sites.” 

The researchers flag that the technique can confirm the safety of food products before they leave the factory and prevent food poisoning.

“One of the most important assessment indicators for ensuring that food is free from contamination is the number of viable bacteria. However, conventional measurement methods take up to two days to yield results, and these results are only available after the food has been shipped from the factory—leading to potentially fatal consequences,” explains Dr. Shiigi.

“Therefore, it is imperative to have a testing method that speeds up the process of identifying bacterial contamination before shipment,” he adds.

According to Dr. Shiigi, the team will continue to optimize the measurement conditions and expect to see the “development of a portable sensor” in line with the development of research aimed at practical applications.

Notably, the method does not require complicated operations or expensive equipment. 

The scientist flags that MTT, a water-soluble molecule, has excellent cell membrane permeability and changes into insoluble reduced formazan inside the cell.

“The number of viable bacteria can be estimated by focusing on the reduction current of MTT remaining in the suspension,” says Dr. Shiigi.

Further, he notes that the standard MTT assay is used as a colorimetric method but requires sufficient incubation time to obtain absorbance. The team found that MTT has electrochemical activity and by “focusing on its current response, highly sensitive measurement became possible.”

The research team has developed the technique based on opinions obtained from interviews with several food product manufacturers in Japan.

“We believe that the product is highly marketable. The plan is to proceed with development with a focus on device realization in the future,” he concludes.

Their results were published in Analytical Chemistry.

Reference:

Journal: Analytical ChemistryTitle: Evaluation of Bacterial Activity Based on the Electrochemical Properties of Tetrazolium SaltsDOI: 10.1021/acs.analchem.3c01871Author: Hikaru Ikeda, Akira Tokonami, Shigeki Nishii, Xueling Shan, Yojiro Yamamoto, Yasuhiro Sadanaga, Zhidong Chen, and Hiroshi ShiigiPublished: August 10, 2023

Abstract

This study focused on the electrochemical properties of tetrazolium salts to develop a simple method for evaluating viable bacterial counts, which are indicators of hygiene control at food and pharmaceutical manufacturing sites. Given that the oxidized form of 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which has excellent cell membrane permeability, changes to the insoluble reduced form of formazan inside the cell, the number of viable cells was estimated by focusing on the reduction current of MTT remaining in the suspension. Dissolved oxygen is an important substance for bacterial activity; however, it interferes with the electrochemical response of MTT. We investigated the electrochemical properties of MTT to obtain a potential-selective current response that was not affected by dissolved oxygen. Real-time observation of viable bacteria in suspension revealed that uptake of MTT into bacteria was completed within 10 min, including the lag period. In addition, we observed that the current response depends on viable cell density regardless of the bacterial species present. Our method enables a rapid estimation of the number of viable bacteria, making it possible to confirm the safety of food products before they are shipped from the factory and thereby prevent food poisoning.

Read more

New Rapid Test for Deadly Mosquito-borne Dengue Virus

University of the Sunshine Coast researchers have developed a rapid portable test for one of the world's fastest-spreading mosquito-borne diseases.

With World Mosquito Day on 20 August marking the ongoing battle against dengue fever in tropical and subtropical countries including northern Australia, the UniSC research team is taking its findings to the next level.

UniSC Associate Professor of Molecular Engineering Dr. Joanne Macdonald published their results in Gates Open Research with co-authors Dr. Madeeha Ahmed and Dr. Nina Pollak.

"We developed a rapid test, with results that look similar to a COVID-19 home stick test, for each of the four types of dengue virus," said Dr. Macdonald. They were sensitive enough to detect even small amounts of viral genetic material in mosquitoes using only pipettes (tubes) and a heating block, instead of expensive laboratory equipment.

"Our entire testing process took about 35 minutes on-the-spot, compared to hours of travel time and PCR processing required for current sampling."

The method involves reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD).

She said the innovative method involved a reagent that inactivated the virus during amplification, enabling simpler, quicker and cheaper detection with a higher level of sensitivity than existing stick tests.

"In practical terms, people and authorities in areas with few resources can set a trap and test mosquitos each week, to check whether dengue is present.

"It has the potential to make mosquito screening more accessible, enhancing surveillance and control efforts in countries where dengue is endemic."

The paper was co-authored by researchers from the QIMR Berghofer Medical Research Institute, Queensland Health and The University of Queensland.

Dr. Nina Pollak has since published a collaborative paper in Microbiology Spectrum investigating the potential of using the tests to detect dengue in human serum, plasma and blood.

Adding co-authors from UniSC (Dr. David McMillan and Malin Olsson), Singapore's National Environment Agency, National University of Singapore, UQ and industry partner BioCifer, this paper also supported the advantages of the new method.

"Our tests provided performance and speed without compromising specificity in human plasma and serum and could become promising tools for the detection of high dengue loads in resource-limited settings," Dr. Pollak said.

The team's next goal is to combine each test for the four dengue serotypes into a single test, to further streamline detection.

Dr. Ahmed said the tests aimed to lay the groundwork for future studies focused on actual use and effectiveness in the field.

"We hope the value of our technology will drive interest among users to conduct field trials in regions where the disease is prevalent," she said.

According to the WHO, dengue fever is a painful and deadly disease that infects up to 400 million people every year. It is a viral infection that spreads to people from mosquito saliva infected with dengue viruses. There is no treatment other than for relief of symptoms, which include high fever, head and body aches, nausea and rash.

More information: 

Madeeha Ahmed et al, Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes, Gates Open Research (2022). DOI: 10.12688/gatesopenres.13534.2

Nina M. Pollak et al, Rapid Diagnostic Tests for the Detection of the Four Dengue Virus Serotypes in Clinically Relevant Matrices, Microbiology Spectrum (2023). DOI: 10.1128/spectrum.02796-22

Read more

Researchers Develop New Rapid and Reliable COVID-19 Detection Method based on MALDI-TOF

Commercially available mass spectrometers can be reliably used to detect the SARS-CoV-2 coronavirus, according to research from the Martin Luther University Halle-Wittenberg (MLU). In a study recently published in Clinical Proteomics, the researchers introduce a novel method that leverages equipment already in use in hospitals and laboratories for detecting bacterial and fungal infections.

The entire process, from taking a swab to receiving results, takes just two hours. The research team believes that this method can be easily adapted to identify other pathogens, potentially serving as a valuable tool in managing future pandemics.

The new method requires a nasal or throat swab. The sample needs to be prepared before it can be analyzed by a mass spectrometer, which takes only a few seconds. In MALDI-TOF mass spectrometry, a laser pulse is used to transfer the sample to the gas phase – then the mass of the individual components is measured.

“This allows us to directly and unambiguously measure individual virus particles of the coronavirus. Thus false-positive results can be ruled out,” says Professor Andrea Sinz from the Institute of Pharmacy at MLU, who specializes in mass spectrometry and proteins. Her team was already able to show in July 2020 that mass spectrometers are generally capable of detecting SARS-CoV-2. However, at this time, the method was still time-consuming and required very high-end equipment.

The advantage of the new method is that MALDI-TOF mass spectrometers are already being used in many laboratories and clinics to diagnose bacterial or fungal infections and are thus readily available. The devices can even distinguish between different variants of the virus. However, the method is not yet as sensitive as polymerase chain reaction (PCR), the most sensitive corona test to date. This means that not all infections may be detected when there is a very low viral load. On the other hand, it is much faster and more flexible.

“In acute phases, the method would make an ideal addition to PCR because we would be able to analyze a lot of samples very quickly. Rapid and reliable results may make it easier to contain outbreaks,” explains Lydia Kollhoff, lead author of the study. Moreover, the approach could be adapted rather easily to other pathogens in future pandemics and supplement PCR testing.

The scientists from Halle want to further optimize the method in partnership with the University of Leipzig Medical Centre. Following this, the method would undergo a certification process so that it could be used clinically.

Reference: 

“Development of a rapid and specific MALDI-TOF mass spectrometric assay for SARS-CoV-2 detection” by Lydia Kollhoff, Marc Kipping, Manfred Rauh, Uta Ceglarek, Günes Barka, Frederik Barka, and Andrea Sinz, 1 July 2023, Clinical Proteomics. DOI: 10.1186/s12014-023-09415-y

The study was funded by the Federal Ministry for Economic Affairs and Climate Action and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation).

Read more

Genie in the LAMP: Rapid Diagnostics Help Protect Horses from Disease Threats

Scientists believe important strides can be made in protecting horses from disease threats, amid growing national and international equine movements, by further developing LAMP technology.

LAMP stands for loop-mediated isothermal amplification. It is a nucleic acid amplification method capable of offering rapid and accurate diagnosis of infectious diseases.

Researchers Alexandra Knox, Gemma Zerna and Travis Beddoe, in a review published in the journal Animals, looked at equine bacterial diseases that pose biosecurity risks and their current diagnostic approaches. For each disease, they looked at developments in terms of LAMP testing.

The trio, with the Department of Animal, Plant and Soil Sciences, part of the Centre for AgriBioscience at La Trobe University in Australia, said the equine industry is an enduring and essential entity worldwide.

It plays a crucial role in many cultures and communities by serving as a source of employment, entertainment, and companionship. It also provides substantial global economic value, estimated at $US300 billion annually.

The authors said the movement and trade of horses have rapidly increased worldwide in recent decades, which has created more opportunities for importation and exposure to diseases. This growth in horse movements has applied pressure on current biosecurity management practices.

“Whilst these measures throughout countries are diligent and ever-adapting to new situations, there remains an apparent urgency for improved surveillance techniques to prevent detrimental disease outbreaks,” they said.

“For a vast number of bacterial diseases of concern, current diagnostics and surveillance methodology rely on out-of-date technology or are time-consuming, with a lengthy turnaround of results.

“In addition to international movement and trade, surveillance at equine events and on farms should remain vigilant; this requires accessible technology that can be utilized in a range of environments, including resource-poor communities.

“Therefore, continuous and rigorous monitoring and detection methods should be of the utmost importance for equine research.”

They said that while attention is largely focused on controlling viral pathogens, bacterial diseases pose similar damaging outcomes.

Many highly contagious bacterial diseases can be transmitted easily from direct contact between horses or indirect contact with contaminated objects, they said.

“Despite extensive biosecurity laws for the importation and exportation of horses worldwide, bacterial outbreaks continue to frequently occur.” Some, they said, are devastating.

“To prevent such events, disease surveillance and diagnosis must be heightened throughout the industry.”

However, current common, or “gold-standard” techniques, have been shown to be inadequate at times. At times, they do not serve the wider community due to the inaccessibility of expensive machinery.

“Additionally, the vast majority of current gold-standard detection methods are time-consuming and do not allow for immediate action in cases of outbreaks.”

Thus, newer technologies are required to impede outbreaks.

“LAMP has proven to be a sound molecular technique that overcomes most pitfalls associated with other nucleic acid amplification techniques,” they said.

Further research into improving LAMP tests has shown promising results that can strengthen this method beyond current capabilities. These include the potential use of additives that have been trialed with other nucleic acid amplification techniques. Additives can increase analytical sensitivity and test stability.

However, as studies for enhancing these tests in equine medicine are limited, the review team strongly recommends further investigations on additive effects for disease detection and surveillance, as it is clear that additive improvements are test-specific.

Furthermore, the use of simple field-deployable amplification and detection techniques has the potential to revolutionize LAMP technology.

“There is an apparent need for rapid results for the implementation of control measures to prevent detrimental spread and outbreaks throughout the equine industry, and thus, it is suggested that the development of LAMP methodologies should be a focal point of research in equine medicine.”

The authors said the robust nature and ease of use of LAMP, coupled with continuous advancements, gives little doubt as to why this technology is being rapidly developed in research for equine disease diagnosis and surveillance.

“Yet, despite numerous assays being designed and optimized for equine bacterial diseases of biosecurity importance, LAMP has failed to replace gold-standard techniques thus far.

“This could derive from a gap in communication between researchers and those who perform diagnostic procedures.”

More advocating regarding the advantages of LAMP, such as the cost and time of such tests, could help overcome this.

“Furthermore, as LAMP is designed to have simplistic and flexible methodologies, thus not requiring trained personnel, there should be more endorsement for those who directly work with horses every day, for example, farmers and stud owners, to self-manage surveillance using these assays (tests).”

Eventually, by using a simple field-deployable “lab-on-a-chip” or microfluidic device, horse owners could potentially be able to perform testing themselves.

“Whilst it is evident that LAMP could become a breakthrough technique for the equine industry, continuous development and optimization do need to occur,” they said.

Additionally, bridging the gap of communication between researchers and diagnosticians regarding LAMP test implementation is essential to advance current diagnostic and surveillance techniques, and continue to protect the equine industry.

Reference:

Knox, A.; Zerna, G.; Beddoe, T. Current and Future Advances in the Detection and Surveillance of Biosecurity-Relevant Equine Bacterial Diseases Using Loop-Mediated Isothermal Amplification (LAMP). Animals 2023, 13, 2663. 

Read more

Tiny Magnetic Beads Produce an Optical Signal that Could be Used to Quickly Detect Pathogens

Getting results from a blood test can take anywhere from one day to a week, depending on what a test is targeting. The same goes for tests of water pollution and food contamination. And in most cases, the wait time has to do with time-consuming steps in sample processing and analysis.

Now, MIT engineers have identified a new optical signature in a widely used class of magnetic beads, which could be used to quickly detect contaminants in a variety of diagnostic tests. For example, the team showed the signature could be used to detect signs of the food contaminant Salmonella.

The so-called Dynabeads are microscopic magnetic beads that can be coated with antibodies that bind to target molecules, such as a specific pathogen. Dynabeads are typically used in experiments in which they are mixed into solutions to capture molecules of interest. But from there, scientists have to take additional, time-consuming steps to confirm that the molecules are indeed present and bound to the beads.

The MIT team found a faster way to confirm the presence of Dynabead-bound pathogens, using optics, specifically, Raman spectroscopy. This optical technique identifies specific molecules based on their “Raman signature,” or the unique way in which a molecule scatters light.

The researchers found that Dynabeads have an unusually strong Raman signature that can be easily detected, much like a fluorescent tag. This signature, they found, can act as a “reporter.” If detected, the signal can serve as a quick confirmation, within less than one second, that a target pathogen is indeed present in a given sample. The team is currently working to develop a portable device for quickly detecting a range of bacterial pathogens, and their results will appear in an Emerging Investigators special issue of the Journal of Raman Spectroscopy.

“This technique would be useful in a situation where a doctor is trying to narrow down the source of an infection in order to better inform antibiotic prescription, as well as for the detection of known pathogens in food and water,” says study co-author Marissa McDonald, a graduate student in the Harvard-MIT Program in Health Sciences and Technology. “Additionally, we hope this approach will eventually lead to expanded access to advanced diagnostics in resource-limited environments.”

Study co-authors at MIT include Postdoctoral Associate Jongwan Lee; Visiting Scholar Nikiwe Mhlanga; Research Scientist Jeon Woong Kang; Tata Professor Rohit Karnik, who is also the associate director of the Abdul Latif Jameel Water and Food Systems Lab; and Assistant Professor Loza Tadesse of the Department of Mechanical Engineering.

Oil and water

Looking for diseased cells and pathogens in fluid samples is an exercise in patience.

“It’s kind of a needle-in-a-haystack problem,” Tadesse says.

The numbers present are so small that they must be grown in controlled environments to sufficient numbers, and their cultures stained, then studied under a microscope. The entire process can take several days to a week to yield a confident positive or negative result.

Both Karnik and Tadesse’s labs have independently been developing techniques to speed up various parts of the pathogen testing process and make the process portable, using Dynabeads.

Dynabeads are commercially available microscopic beads made from a magnetic iron core and a polymer shell that can be coated with antibodies. The surface antibodies act as hooks to bind specific target molecules. When mixed with a fluid, such as a vial of blood or water, any molecules present will glom onto the Dynabeads. Using a magnet, scientists can gently coax the beads to the bottom of a vial and filter them out of a solution. Karnik’s lab is investigating ways to then further separate the beads into those that are bound to a target molecule, and those that are not. “Still, the challenge is, how do we know that we have what we’re looking for?” Tadesse says.

The beads themselves are not visible by eye. That’s where Tadesse’s work comes in. Her lab uses Raman spectroscopy as a way to “fingerprint” pathogens. She has found that different cell types scatter light in unique ways that can be used as a signature to identify them.

In the team’s new work, she and her colleagues found that Dynabeads also have a unique and strong Raman signature that can act as a surprisingly clear beacon.

“We were initially seeking to identify the signatures of bacteria, but the signature of the Dynabeads was actually very strong,” Tadesse says. “We realized this signal could be a means of reporting to you whether you have that bacteria or not.”

Testing beacon

As a practical demonstration, the researchers mixed Dynabeads into vials of water contaminated with Salmonella. They then magnetically isolated these beads onto microscope slides and measured the way light scattered through the fluid when exposed to laser light. Within half a second, they quickly detected the Dynabeads’ Raman signature — a confirmation that bound Dynabeads, and by inference, Salmonella, were present in the fluid.

“This is something that can be used to rapidly give a positive or negative answer: Is there a contaminant or not?” Tadesse says. “Because even a handful of pathogens can cause clinical symptoms.”

The team’s new technique is significantly faster than conventional methods and uses elements that could be adapted into smaller, more portable forms — a goal that the researchers are currently working toward. The approach is also highly versatile.

“Salmonella is the proof of concept,” Tadesse says. “You could purchase Dynabeads with E.coli antibodies, and the same thing would happen: It would bind to the bacteria, and we’d be able to detect the Dynabead signature because the signal is super strong.”

The team is particularly keen to apply the test to conditions such as sepsis, where time is of the essence, and where pathogens that trigger the condition are not rapidly detected using conventional lab tests.

“There are a lot cases, like in sepsis, where pathogenic cells cannot always be grown on a plate,” says Lee, a member of Karnik’s lab. “In that case, our technique could rapidly detect these pathogens.”

This research was supported, in part, by the MIT Laser Biomedical Research Center, the National Cancer Institute, and the Abdul Latif Jameel Water and Food Systems Lab at MIT.

Source: MIT News 

Read more

Clinical Application of Next-Generation Sequencing for Microbiological Diagnosis

Scientists from Belgium have surveyed to understand clinicians’ perspectives on the need for clinical application of next-generation sequencing (NGS) in microbiology laboratories. The study is published in the journal Frontiers in Medicine.

Next-generation sequencing: what are the needs in routine clinical microbiology? A survey among clinicians involved in infectious diseases practice

The survey finds that application of NGS is mostly expected for the diagnosis of neurological and respiratory infections.

Background

The usage of next-generation sequencing (NGS) is increasing firmly in clinical pathology, genetics, and cancer diagnosis. However, the widespread application of this valuable technology as a routine diagnostic tool in clinical microbiology laboratories is still facing major challenges.   

In the field of microbiology, the application of NGS is mostly limited to academic or reference laboratories. The major obstacle against its implementation in clinical microbiology is the lack of standardized protocols or tools.

Major decision-making regarding technologies, operational models, infrastructure, human resources, and professional expertise is needed before the widespread clinical application of NGS.

To facilitate such decision-making, the current survey was conducted among clinicians involved in infectious diseases with the aim of understanding their expectations regarding the added value of NGS for routine clinical care. Another aim of the survey was to identify the factors in which prioritization is needed the most.        

Survey design

This online survey was conducted between January and August 2019 among clinicians practicing in hospitals located in Brussels, Belgium. The survey covered three major topics, including knowledge related to NGS, the expected diagnostic value of NGS, and the expected impact on antimicrobial prescription. 

A total of 24 clinicians completed the survey. Of them, 65.5% were infectious disease specialists, 25% were intensive care specialists, and 12.5% were infectious disease pediatricians.

Survey findings  

The clinician’s knowledge of NGS was analyzed using a scale of 0 to 4, where 0 referred to “none” and 4 referred to “very well.” About 25%, 54.2%, 8.3%, and 12.5% of clinicians rated 0, 1, 2, and 3 on the scale, respectively.

The analysis of answers provided by most of the clinicians in open fields indicated that a wide range of syndromes and samples often remain negative even if there is a strong suspicion of infection.

According to clinicians’ expectations, NGS can provide the highest diagnostic benefit for neurological and respiratory infections, followed by cardiologic and bone and joint infections.

Regarding acute infection sample types, NGS was expected to be beneficial for analyzing cerebrospinal fluid (CSF), pericardial, pleural fluid, and prosthetic materials, as these samples often lack microbiological documentation. Regarding chronic infection sample types, NGS was expected to benefit the analysis of prosthetic materials and bone-derived samples.  

About 83% of clinicians reported considering empirical treatment because of the lack of identification of the exact causative pathogen. Specifically, the survey findings indicated that antibiotics are prescribed blindly in most cases, followed by the prescription of corticoids and antivirals in 40% and 25% of cases, respectively.

All clinicians reported treating patients with low white blood cell counts (neutropenic patients) on a daily basis. About 83% of clinicians reported that identification of a causative pathogen is difficult in these patients. About 46% of clinicians reported that this lack of microbiological diagnosis is due to the lack of sensitivity of routine diagnostic tools.

Significance

The survey evaluates the potential utility of NGS as a routine diagnostic tool in clinical microbiology laboratories. A small group of infectious disease-related clinicians who participated in the survey expect that NGS can potentially improve the quality of microbiological diagnosis, especially for neurological and respiratory infections.

NGS-based identification of actual causative pathogens can prevent the widespread use of empirical treatments, which is a major driving factor for antibiotic resistance.  

Notably, the survey finds a gap between clinicians’ expectations and the actual performance, technical limitations, and lack of interpretability of NGS in clinical microbiology. Thus, more efforts are needed to develop appropriate infrastructure, design routine diagnostic protocols, and involve professional experts for NGS-based microbiological diagnosis.

Journal reference:

Michel C (2023). Next-generation sequencing: what are the needs in routine clinical microbiology? A survey among clinicians involved in infectious diseases practice. Frontiers in Medicine. doi: 10.3389/fmed.2023.1225408. 

Read more

Rapid Infection Test in Dogs Could Curb Antibiotic Resistance

Scientists have developed a new way to rapidly diagnose bacterial infections in dogs, enabling testing and treatment with appropriate antibiotics on the same day.

The method could eliminate the delays associated with conventional diagnosis, in which a sample has to be cultured for days to identify the bacteria present before the appropriate treatment is prescribed.

It is a significant step towards the appropriate use of antibiotics by limiting the use of inappropriate or a wide spectrum of antibiotics for unidentified infections and preventing lengthy courses of treatment.

The development could also be applied across animal and human medicine, for bacterial and other types of infections, researchers say.

New approach

The team used kits optimized for common bacterial species to allow them extract all the DNA from a sample without prior knowledge of which species are present—so-called metagenomic DNA extraction.

They combined this with an existing technology that generates DNA code from samples, known as nanopore sequencing, and a data analysis tool that identifies bacteria according to their DNA fingerprint.

Fast results

This approach allows identification of bacteria in real time, enabling results in a few hours.

The genes identified in the sample also give valuable insight on how the bacteria present are likely to respond to antibiotic treatment, enabling clinicians to prescribe the drug best suited to the infection.

The team tested their system with skin and urinary bacterial infections in dogs, and were able to detect bacteria within five hours.

They were able to identify bacterial species that are difficult to identify with conventional culturing and determine with high sensitivity whether the bacteria present were likely to be resistant to antibiotics.

Wider use

The system is designed to be adaptable for use in various samples and infections across animal species.

In the future it could be useful across a range of animal and human infections, potentially aiding the diagnosis and treatment of other types of infections caused by viruses and parasites, researchers say. The study is published in Microbial Genomics.

"Our method offers a swift way to diagnose bacterial infections and prescribe appropriate antibiotics within hours of patient testing. Following our work with skin and urinary infections in dogs, we are confident that this approach has potential for use across many animal species, and in humans, and has applications in other infection types. It could play a significant role in enabling responsible use of antimicrobial treatments and limiting antimicrobial resistance," says Dr. Natalie Ring.

Reference

Natalie Ring et al, Rapid metagenomic sequencing for diagnosis and antimicrobial sensitivity prediction of canine bacterial infections. Microbial Genomics (2023). 

Abstract

Antimicrobial resistance is a major threat to human and animal health. There is an urgent need to ensure that antimicrobials are used appropriately to limit the emergence and impact of resistance. In the human and veterinary healthcare setting, traditional culture and antimicrobial sensitivity testing typically requires 48–72 h to identify appropriate antibiotics for treatment. In the meantime, broad-spectrum antimicrobials are often used, which may be ineffective or impact non-target commensal bacteria. Here, we present a rapid, culture-free, diagnostics pipeline, involving metagenomic nanopore sequencing directly from clinical urine and skin samples of dogs. We have planned this pipeline to be versatile and easily implementable in a clinical setting, with the potential for future adaptation to different sample types and animals. Using our approach, we can identify the bacterial pathogen present within 5 h, in some cases detecting species which are difficult to culture. For urine samples, we can predict antibiotic sensitivity with up to 95 % accuracy. Skin swabs usually have lower bacterial abundance and higher host DNA, confounding antibiotic sensitivity prediction; an additional host depletion step will likely be required during the processing of these, and other types of samples with high levels of host cell contamination. In summary, our pipeline represents an important step towards the design of individually tailored veterinary treatment plans on the same day as presentation, facilitating the effective use of antibiotics and promoting better antimicrobial stewardship.

Read more