Saturday, May 20, 2023

Plant Science Professors Work with Tech Startup to Create Novel, Rapid Detection of Foodborne Pathogen Test

Recall on lettuce and spinach! These notices have become common across the United States. To protect consumers, produce is routinely tested for foodborne pathogenic bacteria like salmonella, listeria monocytogenes and pathogenic types of E. coli. 

If a plant is infected with plant-based pathogens, the symptoms of infection are easier to observe. It doesn’t work that way with human, foodborne pathogens; you cannot, for example, visibly see E. coli on a plant surface. 

Currently, rapid testing of foods may occur, but it still takes time to figure out who is sick and from where the contaminated product originated. That’s far too late for the many Americans who ate the produce and became sick. The current solution, often a multi-state recall, then becomes damage control. 

University of Delaware researchers want to spot these bacteria before anyone ever falls ill. As detailed in an article published in the Journal of Food Safety, UD and Delaware-based startup Biospection are about to speed up testing — a lot. Faculty members Harsh Bais and Kali Kniel, alongside former graduate student Nick Johnson, teamed up with Andy Ragone of Biospection to detect foodborne pathogens in three to six hours. 

A microbiologist by trade, Kniel is an expert on crossover pathogens like salmonella, which gleefully jump to new hosts like that delicious, fresh lettuce. 

“While the produce industry is working diligently to reduce risks associated with microbial contamination, tools like this have incredible potential to improve risk reduction strategies,” said Kniel, professor of microbial food safety who works regularly with industry and government agencies to reduce risk of foodborne illness. “Collaborations like ours between academics and biotechnology companies can enhance technology and impact food safety and public health.”

These pathogens easily find their way into plants, which are unfortunately very welcoming hosts — hosts that can’t tell you where their guests are. 

Just like humans, plants use defense mechanisms to fight disease. But some human-borne pathogens learned to push open a plant’s open-entry gates called stomates — pores in the leaves or stem — and make themselves at home. 

“Because these bacteria are not true pathogens for plants, you cannot physically see early signs that the plant is under stress,” said Bais, UD professor of plant biology. “Biospection’s technology allows us to say, very quickly, if the opportunistic human pathogen is present in the plant.”

As a chemical physicist working in Wilmington, Ragone got to know Kniel and Bais through Delaware’s scientific community and lab equipment sharing. A relationship built over time, culminating when Kniel, Bais and Ragone applied for and received research funding from a Delaware Biotechnology Institute Center for Advanced Technology (CAT) grant for scientific technology and intellectual property.

The researchers married their interdisciplinary expertise to reduce the risk of foodborne illness, a task that industry and academic researchers struggled with for many years. The result? The team created a multi-spectral imaging platform to look at plant sentinel response. A goal is to use this technique directly on a conveyor, scanning your lettuce before it ever heads to the grocery store.

So how do you see a symptom that you can’t see? The researchers’ technique scans leaves via multispectral imaging and deep UV sensing when the plant is attracting these pathogens. When the researchers looked at benign bacteria, they observed little change. But, with harmful, human-borne pathogens, the test can spot differences in the plant under attack. 

“Using Listeria as an example, in three to six hours, we see a sharp drop of chlorophyll pigments,” Bais said. “That’s a strong signal that the plant is responding physiologically — a marker of unusual bacteria.”

The new, multi-spectral imaging technique is non-invasive, and lightning fast compared to current tests, where a lab scientist extracts a leaf, grinds it up, plates the bacteria and looks for disease. The current method is not commercially available, but Biospection was awarded a National Science Foundation Small Business Innovation Research grant in 2022 to develop and commercialize it into a real time imaging sensor to inspect plants for disease and other stresses. 

“Harsh and Kali were certainly instrumental in the techniques that we developed with multi-spectral imaging and the use of deep ultraviolet fluorescence,” said Ragone, founder and chief technology officer of Biospection. “We built a portable instrument that could be commercialized.”

Vertical farming is an agricultural sector that stands to reap the benefits of this new technology. Using less water and less space, vertical farms are a vital step towards more sustainable agriculture. But when it comes to disease, these farms are just as vulnerable as traditional, outdoor agriculture. An incidence of E. coli means a vertical farm must throw away an entire harvest. 

Biospection is already working with agricultural companies to embed the imaging sensor into vertical farms’ shelves and, for outdoor farms, crop drones. 

“Working with UD, we’ve laid the scientific foundation to create better instruments,” Ragone said. “We’re working toward an instrument that’s portable, automated and can give an answer in a matter of seconds.”

For future research, Bais has his eye on determining if this technology can differentiate between different microbes.

“If the sentinel response is different from one microbe to the other, that gives us the identity of the microbe based on plant sentinel response. We haven’t gone there yet, but that would be the ultimate achievement,” Bais said. “In one sentinel, then you could differentiate between what benign and harmful microbes does this in terms of one sentinel.”

Source: University of Delaware

Saturday, May 13, 2023

Virginia Tech Lab Awarded $1.2 Million to Create Rapid and Accurate Lyme Disease Testing

A rapid, at-home test that can diagnose acute Lyme disease? That is the goal for researcher Brandon Jutras and his team at Virginia Tech’s Fralin Life Sciences Institute.

Through the support of a recent $1.2 million multiyear therapeutic/diagnostic research tick-borne disease grant awarded by the Department of Defense, Jutras’ vision may one day become a reality. This research award aims to improve patient care and quality of life for military service members, veterans, and their beneficiaries as well as the American public living with Lyme disease and other tick-borne diseases.

“Current Lyme disease diagnostic testing is indirect, as it can take weeks, even months, and the results are difficult to interpret, which leads to misdiagnosed or undiagnosed cases,” said Jutras, associate professor in the Department of Biochemistry and an affiliate faculty member in the Center for Emerging, Zoonotic, and Arthropod-borne Pathogens, “It’s an honor to be supported by this innovative program and it is our hope that our work will help former and active service members, their families, and anyone impacted by Lyme disease.”

In developing an acute test to treat Lyme disease, a team of undergraduate and graduate researchers and staff in the Jutras Lab will use peptidoglycan, a component forming the cell walls of many bacteria, as a biomarker of acute disease.

“Peptidoglycan is a very abundant molecule that’s naturally being shed by the bacterium,” Jutras said. “And most importantly, when compared with other bacteria, this molecule is extremely unique to the bacterium that causes Lyme disease. And so we are developing a sophisticated but very accessible test that can exploit these unusual molecular signatures to directly detect this molecule and in essence be able to hopefully diagnose Lyme disease within hours after infection.”

Since it was first identified in the United States in 1975, Lyme disease has become the world’s most common tick-borne zoonotic disease — one spread from animals to humans through the bite of infected ticks —  according to the Centers for Disease Control and Prevention. In fact, more than 14 percent of the global population is thought to currently have Lyme disease or have been previously infected. The congressionally directed program to support fundamental research on tick-borne disease was established in 2016.

The rapid and accurate diagnosis of this disease is a priority in patient care so as to avoid the medical consequences associated with delayed treatment. It also could help uncover the true prevalence of Lyme disease, which can be difficult to determine because of problems associated with current diagnostic testing.

“These Lyme disease tests are not only insufficient, but they also lack the two main requirements for any diagnostic test – sensitivity and specificity,” said Osamudiamen Ebohon, a graduate student that matriculated into the Jutras Lab through the center’s new Interdisciplinary Graduate Education Program in Infectious Disease. “In the long run, this may have an impact not only on diagnosis but also on monitoring the spread of the disease and the development of appropriate interventions.”

The success of the labs’ new approach is the recent creation of several specific monoclonal antibodies that can detect the unique pieces of peptidoglycan, efforts that were partially supported by an award from the Bay Area Lyme Foundation.

“Whenever we tackle a complex problem, we always take a multisystem approach. Support from the Bay Area Lyme Foundation provided the freedom to explore and in doing so, lead to an exciting discovery — the creation, production, and characterization of an entirely new set of monoclonal antibodies which were the basis of the Department of Defense award and may be a game-changer for diagnostic and maybe even therapeutic purposes,” said Jutras.

Concurrent to the grant-funded research, Jutras’ exploration into developing an accessible test is augmented with the support of the U.S. Department of Health and Human Services and the Steven & Alexandra Cohen Foundation LymeX Diagnostics Prize.

Jutras, along with colleagues Richard Helm, associate professor of biochemistry in the College of Agriculture and Life Sciences at Virginia Tech, and Marcos Pires, associate professor in the Department of Chemistry at University of Virginia, are currently in the second phase of the competition, which coincides with the next phase of federal nurturing of tick-borne-disease solutions.

With this new phase, which runs through September, $10 million in LymeX prizes are projected to be available across all potential competition phases, subject to availability and approval of funds.

MSU Partners with Physician in the Dominican Republic to Improve Tuberculosis Testing

Tuberculosis, or TB, a bacterial disease that usually attacks the lungs, has regained its distinction as the leading infectious disease resulting in death, following the extraordinary efforts to curb the spread of COVID-19. The bacteria that cause tuberculosis, Mycobacterium tuberculosis, are spread through the air, for example when someone with the disease coughs or sneezes. The disease kills nearly 1.5 million people each year.

Early and widespread detection of TB is crucial to slow its spread and save lives, according to Robert Paulino-Ramírez, M.D., principal investigator at the Institute of Tropical Medicine & Global Health at the Universidad Iberoamericana in Santo Domingo, Dominican Republic, and chair of the Tropical Medicine/Infectious Disease Virtual Institute in the Education and Research Consortium of the Americas within the Michigan State University College of Osteopathic Medicine Institute for Global Health.

“In the Dominican Republic, the tuberculosis rate is very high,” Paulino-Ramírez said. “One of the most important gaps in treatment is rapid identification of active cases at the community level.”

To recognize community transmission requires rapid diagnosis, but currently the only way to do that in the Dominican Republic national health care system is through Polymerase Chain Reaction, or PCR testing, a time-consuming and expensive procedure requiring highly trained technicians. “PCR testing is cost-prohibitive and even the reagents required are in short supply due to supply chain issues,” said Paulino-Ramírez.

A $142,000, two-year grant from the Dominican Ministry of Higher Education, Science and Technology will allow Paulino-Ramírez, in partnership with Michigan State University, to leverage expertise and new technologies to alleviate that problem.  

A crucial part of the solution comes from the Nano-Biosensors Lab of Evangelyn Alocilja, Ph.D., professor in the MSU Department of Biosystems and Agricultural Engineering, and Ruben Kenny Briceno, M.D., IGH Co-Coordinator Peru Research and member of the Nano-Biosensors Lab, who developed a test for TB that uses nanoparticles targeted to specific proteins and genes of the tuberculosis bacteria.

The test is about 30 times cheaper than PCR, requires very little training time for health care personnel and even students, and, unlike the reagents used in PCR testing, can be stored at room temperature, according to Dr. Paulino-Ramírez. “The technology has been demonstrated to perform extremely well in vitro.”

“The nanoparticle-based TB test has been validated in hundreds of sputum samples in Mexico, Peru and Nepal, and the results are comparable to the PCR-based GeneXpert system,” Alocilja said. “Dr. Paulino-Ramirez and I are excited to work together toward potentially improving TB diagnosis in the Dominican Republic.”

How the body reacts to infection depends greatly on the health of a person’s immune system. Therefore, the team will first confirm the efficacy of the nanoparticle test vs. PCR in a clinical setting for both immune-suppressed and non-immune-suppressed patients. If confirmed to be effective, “the transfer of this technology will be hugely beneficial, especially in middle- and lower-income countries,” Paulino-Ramírez said. Currently, patients are often treated based on symptoms alone rather than a positive TB test, simply because the cost is too high or the materials are unavailable.  

“Without the need for costly molecular biology laboratories and -70°C storage facilities (both are required for PCR), it will be much easier for health care providers at the community level to detect TB,” Paulino-Ramírez said.

Paulino-Ramírez is excited about the potential of the new technology and its ability to expand disease detection. Because nanoparticles are easy to tailor to specific diseases, Paulino-Ramírez, Alocilja, Briceno and the collaborating team are optimistic that the technology can be adapted in the future to detect other diseases, including coronaviruses.

“The Institute for Global Health and the College of Osteopathic Medicine are privileged to partner with the College of Engineering and the Tropical Medicine Institute at UNIBE, Dominican Republic, to expand research on early diagnosis of tuberculosis and other infectious diseases through nanotechnology. Dr. Briceno de la Cruz from IGH, Dr. Alocilja from COE, and Dr. Robert Paulino, main researcher at the Tropical Medicine Institute, will lead the next phase of this project in the Dominican Republic," said William Cunningham D.O., MHA, director of IGH and associate dean for Global Health at MSUCOM.

A Smoke Detector for Viruses

The COVID-19 pandemic spiked anxiety about an invisible threat: airborne viral particles. The inability to detect the presence of these microscopic pathogens necessitated the disruptive precautionary measures of the COVID-19 era, such as masks, social distancing and the shutdown of schools, workplaces and large public gatherings. 

But what if airborne viruses could be detected in the same manner as smoke, carbon monoxide and other environmental dangers? Two University of Illinois Chicago scientists have collaborated on a device that could detect SARS-CoV-2, influenza, RSV and other pathogens. The technology, called BioAerium, could dramatically improve disease surveillance for public health as well as research on how viral particles move through the air.  

The device earned its creators Michael Caffrey, professor of biochemistry and molecular genetics, and Igor Paprotny, associate professor of electrical and computer engineering, the 2022 Inventor of the Year award from UIC. Their work is also the basis of a new startup company, also called BioAerium, which is exploring commercial opportunities of the technology.  

While COVID-19 accelerated the urgency of Caffrey and Paprotny’s project, their collaboration actually formed before the pandemic, with the flu virus as their initial target. Caffrey’s research studies the structure of viruses such as influenza, HIV and Ebola using laboratory methods to reveal the intricate architecture of these pathogens and how it relates to their activity. Paprotny’s expertise is in the study and design of microfluidic systems, which work with tiny amounts of fluid, usually liquid; however, most of his devices work to detect aerosols – minute particles in the air.  

Because a respiratory virus detector needs to detect small quantities of viral bioaerosols — aerosol that contain viruses — the combination of their specialties provided the ideal partnership for the challenge. 

“The viruses that I study are, for the most part, airborne,” Caffrey said. “Viruses in air form larger particles, and those particles are analogous to many of the particle types that Igor’s lab was studying before. So, it was a natural fit.” 

Devices for detecting airborne viruses already exist, but they are bulky and expensive, making them impractical for widespread use. The technique for identifying a target virus, DNA amplification, is traditionally performed in a laboratory, so that step needs to be both automated and miniaturized in order to create a portable or wearable detector.  

Recent “lab-on-a-chip” technologies – similar to what makes a home COVID-19 test work – address this challenge, but typically require high-concentration samples from saliva or a nose swab. So Caffrey and Paprotny needed to tackle two challenges: shrinking down the biochemical analysis and collecting enough viral particles from the air for it to work.  

Enter the UIC nanotechnology core facility, where Paprotny is the faculty research director. With microfabrication – the manufacturing of complex technologies at an exceedingly small size – the researchers could design detectors that are both practical for non-laboratory use and inexpensive enough to make at scale 

“We’re in this nice sweet spot where we had a lot of technology that was already developed on the air microfluidic side, and we connected with Mike who was an expert on the virology side,” Paprotny said. “By connecting the two together, we could come up with this device that’s really novel and has a lot of promise going forward.” 

Currently, the BioAerium prototype detects one virus at a time; for example, a detector could be set up to look for SARS-CoV-2 and placed in a classroom or airplane to monitor the air for the virus that causes COVID-19. But Caffrey and Paprotny designed the device as an open, customizable platform and envision a future “multiplex” version that can detect many viruses at once, or even distinguish between variants of a virus. 

“COVID is maybe going away, but we’re looking at this project as preparing for the next pandemic, and for diseases like the flu where it will be beneficial to be able to detect the presence of a virus,” Paprotny said. “We may even be able to tell whether we are detecting a flu variant that people are vaccinated against or a different strain. Making those distinctions can be especially important.” 

The small size and low cost of the device could also enable exciting new science. With multiple detectors installed in a building, across a campus, or throughout a neighborhood, researchers could study in unprecedented detail how a virus spreads through the air and detect emerging variants in real-time, instead of waiting for infections and patient tests. 

“We envision that such devices would be connected to the Internet of Things,” Caffrey said. “One could then take advantage of big data science to analyze the signals coming out in multiple areas and provide a global picture that would be useful from a public health perspective.” 

Caffrey and Paprotny are currently working with the Office of Technology Management on commercializing their detector technology and have filed for patents. For more information, visit the BioAerium website. Read more about the Research, Scholar, and Inventor of the Year award recipients at the Office of the Vice Chancellor of Research website.

A COVID-Detecting Breathalyzer Utilizing Laser Technology

Scientists from CU Boulder and the National Institute of Standards and Technology (NIST) have developed a laser-based breathalyzer powered by artificial intelligence (AI) that can detect COVID-19 in real-time with excellent accuracy. The device uses breath analysis as an alternative, rapid, non-invasive test for COVID-19 and has the potential to diagnose diverse conditions and disease states. The “frequency comb breathalyzer” uses Nobel Prize-winning technology from CU and could revolutionize medical diagnostics.

Scientists from CU Boulder and the National Institute of Standards and Technology (NIST) have developed a laser-based breathalyzer powered by artificial intelligence (AI) that can detect COVID-19 in real-time with excellent accuracy. The device uses breath analysis as an alternative, rapid, non-invasive test for COVID-19 and has the potential to diagnose diverse conditions and disease states. The “frequency comb breathalyzer” uses Nobel Prize-winning technology from CU and could revolutionize medical diagnostics. The team is now focusing on a wide range of other diseases with the potential for patients to blow into a mouthpiece integrated into their phones to get real-time health information.

The breathalyzer uses the unique chemical fingerprint or “breathprint” that humans exhale with each breath. It produces over 1,000 distinct molecules that can provide valuable insights into what’s happening inside the body. For years, scientists have tried to harness this information, using dogs, rats, and even bees to sniff out diseases such as cancer, diabetes, and tuberculosis.

The multidisciplinary team of physicists, biochemists, and biologists is now focusing on a wide range of other diseases, hoping that the “frequency comb breathalyzer” could revolutionize medical diagnostics. The breathalyzer was born of Nobel Prize-winning technology from CU and has the potential to diagnose diverse conditions and disease states rapidly and non-invasively.

“Our results demonstrate the promise of breath analysis as an alternative, rapid, non-invasive test for COVID-19 and highlight its remarkable potential for diagnosing diverse conditions and disease states,” said Qizhong Liang, a PhD candidate in JILA and the Department of Physics at CU Boulder, and the first author of the study.

The team is hopeful that in the future, people could go to the doctor and have their breath measured alongside their height and weight. Alternatively, they could blow into a mouthpiece integrated into their phone and get real-time information about their health.

Since then, Ye’s team has improved the sensitivity of the technology a thousandfold, enabling the detection of trace molecules at the parts-per-trillion level. They have also linked specific molecules to disease states, paving the way for the breathalyzer’s potential use in medical diagnostics.

The team’s findings were published in the Journal of Breath Research on April 5. The research is a result of a collaboration that began during the COVID-19 pandemic.

The breakthrough represents a significant step forward in the diagnosis of diseases using exhaled breath. The breathalyzer’s potential to diagnose COVID-19 and other diseases rapidly and non-invasively could revolutionize medical diagnostics, making it possible for people to get real-time information about their health. The potential of the breathalyzer is endless, and the team is hopeful that it will change the face of medical diagnostics in the future.

Reference: Liang Q, Chan YC, Toscano J, et al. Breath analysis by ultra-sensitive broadband laser spectroscopy detects SARS-CoV-2 infection. J Breath Res. 2023;17(3):036001. doi: 10.1088/1752-7163/acc6e4

Monday, May 1, 2023

Chula Researchers Develop a Rapid MTB Strip Test for Tuberculosis

Lecturers of the Faculty of Allied Health Sciences, Chulalongkorn University have developed MTB Strip Test Kit for Tuberculosis (TB) diagnosis that’s accurate and easy to use, guaranteed by the 2023 Invention Award from the National Research Council of Thailand (NRCT) — Another hope to reduce the spread of tuberculosis in Thailand.

Tuberculosis is one of the most contagious diseases that continues to challenge the public health system today. Although the World Health Organization (WHO) aims for 2035 (the next 12 years) to be the year to end the global tuberculosis crisis, the disease trend is still worrisome.

“Thailand is one of the 14 countries with the most severe TB incidence. Fortunately, drug-resistant tuberculosis in Thailand has been removed from the WHO’s list of highest-incidence countries. Only ordinary tuberculosis cases remain,” said Associate Professor Dr. Panan Ratthawongjirakul, Department of Transfusion Medicine, Faculty of Allied Health Sciences, Chulalongkorn University, discussing the situation of tuberculosis in Thailand.

Tuberculosis is an airborne disease caused by a bacterium called “Mycobacterium tuberculosis”. It is spread from TB patients to others through small respiratory secretions (AKA droplets) that come from coughing, sneezing, or talking. It is easy to contract and it spreads quickly. 

“One of the mechanisms to help end tuberculosis is identifying TB patients as early as possible to control and limit its transmission” said Assoc. Prof. Dr. Panan about the inception of the research project to develop MTB Strip (Mycobacterium tuberculosis Strip) that is easy to use, convenient to read by the naked eye, and with fast and accurate results. More importantly, the cost should not be high to make it accessible to local public health service systems.

“If we can distribute this test to small hospitals everywhere, we will be able to identify TB patients within two hours and screen positive patients quickly into the treatment system.  We believe this will help reduce the number of TB cases in our country” said Assoc. Prof. Dr. Panan about the objective of MTB Strip innovation.

Pros and Cons of the current methods of TB Testing

Assoc. Prof. Dr. Panan mentioned the various advantages and disadvantages of current testing methods for tuberculosis as follows:

1. Microscopic examination using acid-fast staining is a simple method. It can be done in a small hospital, but the disadvantage is low sensitivity (the minimum bacterial concentration required for a positive signal when examining with a microscopic examination is 5000–10000 cells in 1 ml of sputum.

2. Sputum culture is the standard method of diagnosing tuberculosis, but it can only be done in well-equipped large hospitals. This method must be done in a room with a high-safety system to prevent it from spreading outside. It takes more than a month to know the results which will result in delayed treatment.

3. TB Genotyping involves taking the patient’s sputum to extract and amplify the genetic materials which are then tested by a Real-time PCR machine. The disadvantage of this method is that it is costly and requires a lab with specialized personnel, so it can be done only in some hospitals.

Based on the advantages and limitations of various methods used to detect tuberculosis, the research team developed the MTB Strip Test Kit.                         

Faster and easier TB Screening with MTB Strip

MTB Strip TB Test Kit consists of 2 main parts: 1. Genetic amplification using isothermal amplification with specifically modified and designed primers. 2. Genetic materials detection using developed test strips, which are manufactured from ISO13485-certified industrial plants for medical device manufacturing.

Assoc. Prof. Dr. Panan explained the process of using this test kit “after receiving sputum from the patient, the DNA will be extracted and used as a template. We will put a primer specially designed to amplify the amount of genetic material in the DNA of the pathogen in the patient’s sputum before entering the isothermal amplification process by using a recombinase polymerase amplification technique. It takes only 20 – 40 minutes at 37 degrees Celsius. Then, the developed test strip is dipped into the amplified genetic material. The results will appear on the test strip as positive and negative results like the ATK test that we are familiar with.”

The key feature of the MTB Strip is its sensitivity to tuberculosis. With a small amount of tuberculosis in the sputum, the test can detect it and display the result. In addition, the test process takes less than an hour and does not require any special tools.

“The results are up to 96 percent accurate compared to Realtime PCR and other commonly used acid-resistant dye methods. Importantly, this kit is cheaper than molecular biology tests because it does not require any special tools such as thermocycler” Assoc. Prof. Dr. Panan emphasized.

The MTB Strip kit uses the principle of amplifying genetic material under a single constant temperature in conjunction with a heat box. In a typical laboratory, this type of box is already available. Small hospitals can also use this technique.

“The MTB Strip TB test kit we have developed will enable many existing small and medium-sized hospitals in Thailand to screen for TB cases so that patients can receive appropriate treatment quickly, thereby reducing the number of TB cases and the spread of TB.”

Fighting tuberculosis with the Distribution of MTB strips to the provinces

The MTB Strip Test prototypes have already been administered at Umphang Hospital, Tak Province in 2019-2020 and the results are good to a certain extent. However, Assoc. Prof. Dr. Panan has not stopped developing methods and innovations to reduce the number of cases of tuberculosis in Thailand.

“Although the MTB Strip kit works satisfactorily, we would still like to develop more sensitivity by making the DNA extraction easier to be used as the kit primer.”

In addition, Assoc. Prof. Dr. Panan also has plans to expand the testing of TB and related diseases by developing an easier-to-use DNA extraction kit and TB test kit that can identify drug-resistant variants of TB right from the outset, so that more specific treatment guidelines can be set.

“We are currently conducting in-depth research on the genetic modification of tuberculosis using a novel technique of genetic modification for a living organism called CRISPR Cas-9 Interference to modify certain TB genes, making the infection less aggressive and more responsive to antituberculosis drugs. CRISPR Cas-9 Interference can be used in conjunction with current antituberculosis drugs.”

If the study is successful, it will be a new TB treatment of the future, which Assoc. Prof. Dr. Panan is sure will help reduce the number of TB cases to reach WHO’s target. Small hospitals interested in the MTB Strip Test kits can contact Assoc. Prof. Dr. Panan Rathwongjirakul, the Research Unit of Innovative Diagnosis of Antimicrobial Resistance, Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, email

Early Pathogen Detection: Collaboration Speeds up Bioelectronic Sensor Development

“The key to successful collaboration with clinicians is to spend time with them, getting to know exactly what they need,” said Sahika Inal, bioengineer at KAUST. “Creating tools for doctors to use at the point of interaction with patients requires full understanding of healthcare workflows and the expertise of the workforce. If you don’t have this, then the tools are likely to be ignored.”

Inal and her team are collaborating closely with Ashraf Dada, Fatima Alhamlan and co-workers at King Faisal Specialist Hospital and Research Centre (KFSH&RC) in Saudi Arabia. The aim of the partnership is to help develop and trial bioelectronic sensors that will aid in cheap, accurate and rapid pathogen detection.

“My aim is to make doctors’ jobs easier and diagnosis as fast as possible by providing a new technology to replace conventional laboratory tests,” Inal said. “Our goal is to make these sensors available to clinicians so that they can get data to diagnose diseases faster. We’re also hoping that the technology will support healthcare professionals in low-income countries and in communities remote to healthcare services.”

During the pandemic, Inal joined forces with KAUST’s Stefan Arold to develop electronic chips that can detect the presence of COVID-19 from saliva samples. Their chips are close in sensitivity to conventional PCR tests and provide results in just 15 minutes.

“To investigate this innovative technology for its suitability in a clinical setting and to validate the accuracy of our sensors, we reached out to experts at Saudi Arabian hospitals,” Inal said.

Researchers from KFSH&RC provided us with samples and evaluated the results based on their conventional techniques as a comparison tool. They then shared their results with us, enabling us to validate our technology. This is how our collaboration started.”

“Our hospital has an advanced Research Centre to support the clinical health care of our patients with innovative diagnostics and therapeutic studies,” Dada said. “We were delighted with the novelty, sensitivity and accuracy of the diagnostic approach brought to us by KAUST researchers.”

Since their trials for the COVID-19 sensors, the collaboration between Arold, Inal and the hospital teams has gone from strength to strength, with the teams working closely to expand the potential of the bioelectronic sensors.

“The clinicians make our research relevant – they tell us what is really missing in their daily routine, which tools they would have liked to have,” Inal said.

“Understanding this then benefits both physicians and patients because conditions can be treated more rapidly. Our devices will allow healthcare providers to screen for multiple markers in a short time, allowing them to build a clearer picture of each patient’s overall health. From our perspective, being able to validate our sensors using high-quality authentic data that we know has been collected with care is invaluable.”

“I hope that our project will result in a cutting-edge technology that revolutionizes the diagnostics of pathogens and changes the landscape of diagnostic tools in the field of infectious diseases,” Dada said. “Such technology should also help ensure that the world is better prepared for future pandemics.”

Inal hopes that their technology will advance rapidly to provide early, accurate detection for both infectious and noninfectious diseases. Both Inal and Dada are excited to see the fruits of this collaboration rolled out more widely in future. 

Tuesday, April 25, 2023

Testing Antibiotic Resistance with a Fast, Inexpensive and Easy Method

Researchers at EPFL and Vrije Universiteit Brussel have developed a novel and highly efficient method for rapid antibiotic susceptibility testing using optical microscopy. The technique, called Optical Nanomotion Detection, is extremely rapid, single-cell sensitive, label-free, and requires only a basic traditional optical microscope, equipped with a camera or a mobile phone.

“We have developed a technique in our laboratories that allows us to obtain an antibiogram within 2-4 hours – instead of the current 24 hours for the most common germs and one month for tuberculosis,” says Dr Sandor Kasas at EPFL. Professor Ronnie Willaert at Vrije Universiteit Brussel adds: “Our technique is not only faster but also simpler and much cheaper than all those existing now.”

Antibiotic resistance happens when bacteria develop the ability to defeat the drugs designed to kill them. It has now grown into a global public health issue. It was responsible for at least 1.27 million deaths worldwide in 2019 while being involved in nearly five million deaths. Every year, the US sees almost three million antimicrobial-resistant infections, with the cost of treating the six most common ones at over $4.6 billion. The EU sees almost 700,000 cases each year, which cost it an estimated €1.5 billion.

Antibiotic sensitivity testing (AST) uses culture methods that expose bacteria to antibiotics, or genetic methods to determine if bacteria possesses genes that confer resistance. Typical ASTs last up to 24 hours or even longer for slow-growing bacteria – a timeframe that can mean life or death in a clinical setting. There have been some faster ASTs developed in recent years, but they tend to be complex, needing sophisticated and expensive equipment.

Now, researchers led by Kasas and Willaert have developed a fast, cheap, and widely accessible method based on optical microscopy that can perform an AST with single-cell sensitivity and without needing to attach or label bacteria. The technique uses a basic, conventional optical microscope, a camera or mobile phone, and dedicated software. The joint research project is published in PNAS.

The new technique is called optical nanomotion detection (ONMD), and involves the monitoring of nanoscale vibrations of single bacterial before and while being exposed to antibiotics. The monitoring is performed with a basic optical microscope, a video camera or a mobile phone.

The ONMD technique monitors the microscopic oscillations of bacterial cells (nanomotion) that characterize living organism and can be considered as a “signature of life”. Indeed, nanomotion lasts as long the organism is alive but stops immediately when it is dead. In the ONMD technique, bacterial nanomotion is recorded in a movie in which all individual cell displacements are monitored with sub-pixel resolution.

The researchers used ONMD to successfully detect the sensitivity of numerous bacteria to antibiotics. Escherichia coli, Staphylococcus aureus, Lactobacillus rhamnosus, and Mycobacterium smegmatis (a non-pathogenic bacterial model for tuberculosis) sensitivities to the antibiotics ampicillin, streptomycin, doxycycline, and vancomycin was determined in less than two hours.

The ONMD not only monitors the bacteria life-death transitions upon exposure to different antibiotics but also highlights changes in the bacteria’s metabolism caused by the availability of nutrients. The tests showed that ONMD can assess the sensitivity or resistance of bacterial cells to antibiotics in a simple and rapid way by monitoring cellular oscillations.

The authors state: “The simplicity and efficiency of the method make it a game-changer in the field of AST” as it can be applied to a wide range of bacteria, which has significant implications for clinical and research applications.

Other contributors

University of Lausanne (UNIL)


Swiss National Science Foundation (SNSF)

Research Foundation – Flanders (FWO)

Belgian Federal Science Policy Office (Belspo)

European Space Agency (ESA)


Maria I. Villalba, Eugenia Rossetti, Allan Bonvallat, Charlotte Yvanoff, Vjera Radonicic, Ronnie G. Willaert, Sandor Kasas. Simple optical nanomotion method for single-bacterium viability and antibiotic response testing. PNAS 28 April 2023. DOI: 10.1073/pnas.2221284120 

Source: École Spéciale de Lausanne

Tuesday, April 18, 2023

Researchers Develop Rapid Test for Salmonella Food Contamination

McMaster University researchers have developed a rapid and inexpensive test for Salmonella contamination in chicken and other food — one that’s easier to use than a home COVID test.

The test, described in a new paper in the journal Angewandte Chemie, could improve food safety, reduce the cost of processing fresh poultry and other foods, and help to limit broad recalls to batches that have specifically been identified as contaminated.

The researchers have shown that the test provides accurate results in an hour or less without the need for accessories or a power source, compared to today’s monitoring through lab cultures, which require at least a full day to produce results.

Once scaled up and made available commercially, the new test could be a significant boon to poultry processors, for whom salmonella is among the most significant contamination risks.

The test would also be beneficial for ensuring the safe processing of other foods that are particularly vulnerable to salmonella, such as eggs, dairy products and ground beef.

A single major poultry processor performs tens of thousands of salmonella lab tests each year. Reducing or even eliminating the need for overnight lab cultures would represent significant savings and make it easier to identify contamination earlier in the process.

“Anyone can use it right in the setting where food is being prepared, processed or sold,” says study co-author Yingfu Li, a professor of Biochemistry and Chemical Biology who leads McMaster’s Functional Nucleic Acids Research Group.

Protecting the public from salmonella is a high priority for food producers, retailers and regulators alike, since it is one of the most common and serious forms of food-borne infection, causing 155,000 deaths globally every year.

What makes the test work is a new synthetic nucleic acid molecule, developed at McMaster. For the test, the molecule is sandwiched between microscopic particles such as gold.

The test platform lines the inside of the tip of a pipette and begins to work when a liquefied sample of the food being tested is drawn inside the tube.

If salmonella bacteria are present, they cut through the particles, allowing the molecule to escape.

When the solution is dropped onto a paper test strip, the presence of salmonella shows as a visible shade of red, thanks to a new form of biosensor, also created by the McMaster team. The greater the concentration of salmonella, the brighter the colour appears.

The new technology has been developed with support from the non-profit research organization Mitacs, and Toyota Tsusho Canada Inc., an indirect subsidiary of Toyota Tsusho Corporation in Japan, which plans to develop the innovation for commercial use.

The research is part of an ongoing, broader effort to establish McMaster and its Global Nexus for Pandemics and Biological Threats as a centre for the development of real-time sensors, pathogen-repellent materials and other products that improve food safety.

“This is very important to us in the development of our food-testing program,” says co-author Tohid Didar, an associate professor of Mechanical Engineering and Canada Research Chair in Nano-biomaterials.

Li, Didar and Filipe authored the paper with postdoctoral research fellow Jiuxing Li, PhD student and Vanier Scholar Shadman Khan, and research associate Jimmy Gu.

Reducing illness and food waste aligns with Toyota Tsusho Canada’s values, explained Toyota Tsusho Canada Inc. President Grant Town.

“Our goal is to help bring proven research from the lab to the marketplace, where it can benefit society,” Town says. “Reducing the risk of illness while also cutting food waste will benefit everyone, and Toyota Tsusho Canada sees this as a great opportunity.”


A Simple Colorimetric Au-on-Au Tip Sensor with a New Functional Nucleic Acid Probe for Food-borne Pathogen Salmonella typhimurium. Li, J., Khan, S., Gu, J., Filipe, C. D. M., Didar, T. F., Li, Y., Angew. Chem. Int. Ed. 2023, e202300828; Angew. Chem. 2023.

Saturday, April 15, 2023

Microbiology Poems, Courtesy of ChatGPT

ChatGPT is a natural language processing tool driven by AI technology that allows you to have human-like conversations and much more with the chatbot. The language model can answer questions and assist you with tasks like composing emails, essays, and code.

I was curious as to the power of using ChatGPT to write an essay about microbiology. But to have fun, I asked the chatbot to write poems about microbiology and more specifically, about rapid microbiological methods. Within 5 seconds, surprisingly accurate (and humorous) poems were written, which I have reproduced below. My disclaimer: ChatGPT must obtain its information from somewhere in public domain (e.g., Wikipedia, books, news articles, scientific journals), and as such, the poems presented below may have been generated from prior published work. 

A Poem About Rapid Microbiological Methods (my favorite)

In labs of science, with cutting-edge tech,

Rapid microbiological methods, a breakthrough, a check,

A faster way, a modern stride,

To detect and identify, with precision, worldwide.

Gone are the days of lengthy waits,

For microbial results, sealing fates,

Rapid methods, swift and true,

Reducing time, a remarkable coup.

PCR, MALDI-TOF, and more,

Using automation, they soar,

High-throughput screening, in a flash,

Rapid methods, a time-saving splash.

From food safety to clinical care,

Rapid methods, a boon, so rare,

Detecting pathogens, with speed,

Saving lives, meeting the need.

In pharmaceuticals, in water, in air,

Rapid methods, with accuracy, they dare,

To identify contaminants, in a blink,

Empowering industries, with a new link.

Quality control, process monitoring,

Rapid methods, an efficient adorning,

Ensuring safety, reducing risk,

Microbial analysis, at a brisk.

But validation, a crucial part,

Ensuring reliability, from the start,

Robustness, accuracy, and precision,

Validating rapid methods, a mission.

So, here's to rapid microbiological ways,

Advancing science, in modern days,

Bringing speed, efficiency, and control,

Rapid methods, a boon to behold.

A Poem About Microbiology (#1)

Microbes unseen, a world so small,

Invisible to the naked eye, yet rule them all.

They dwell in the air, in water, in soil,

Microbes in every corner, a hidden coil.

Bacteria, fungi, viruses, and more,

Microbiology's wonders to explore.

They're the masters of decomposition,

Breaking down the organic with precision.

In labs, scientists study their ways,

Peering through microscopes, in a curious gaze.

Culturing, identifying, studying their traits,

Unraveling mysteries, their intricate debates.

From penicillin to vaccines and beyond,

Microbes offer solutions, a treasure to be found.

They can cure, they can harm,

Infectious diseases can cause alarm.

But microbiology is a field of great might,

Harnessing nature's power, shining bright.

Understanding the tiny world of life,

Bringing knowledge, reducing strife.

In food, in health, in the environment,

Microbes play a role, so significant.

From fermentation to biotechnology,

Microbiology's impact, a boundless sea.

So let us marvel at the unseen,

The microbial world, a wonder so keen.

A realm of diversity, complexity,

Microbiology's beauty, a true delicacy.

A Poem About Microbiology (#2)

In a microscopic world unseen,

Where wonders dwell, both small and keen,

Microbes thrive, in vast array,

A realm of life, in a tiny display.

Bacteria, tiny cells, so diverse,

In shapes and sizes, they immerse,

Some are round, some are rod-like,

In a microscopic world, they strike.

Viruses, smaller still, but mighty,

Intracellular hijackers, acting slyly,

Invading host cells, causing disease,

Microbiology's puzzle, a complex tease.

Fungi, with their branching threads,

Mycelium network, like nature's threads,

Mushrooms, molds, and yeasts, they grow,

A world of fungi, both friend and foe.

From gut to skin, from air to soil,

Microbes thrive, in their daily toil,

They decompose, they ferment,

In symbiosis, they are inherent.

They aid digestion, break down waste,

In bioremediation, they show their taste,

In brewing, baking, and making cheese,

Microbes play a role, with utmost ease.

In labs, scientists study their ways,

Unraveling secrets, through endless days,

Identifying species, understanding their traits,

Microbiology's mysteries, with endless rates.

They create antibiotics, vaccines, and more,

Innovations galore, a microbial store,

Solving problems, finding cures,

Microbes, a blessing that endures.

So let's appreciate the microscopic view,

The world of microbiology, a wonder so true,

A realm of life, both small and grand,

Microbes, a marvel, forever to stand.

Label-free Droplet-based Methods Improve Rapid Screening and Sorting of Bacteria

Effective, accurate and quick ways to screen and sort microbes are in short supply. Most methods available now rely on additional labeling steps to sort bacteria, which are typically time-consuming and cannot work well for industrial-scale breeding. 

The need for accuracy and speed are met using a label-free droplet-based integrated microfluidic platform that screens bacterial growth based on phenotype, or observable characteristics. Better and quicker screening of bacterial growth can have considerable effects on medical, pharmaceutical and agricultural industries. 

Screening of bacteria by their observable characteristics may not seem possible, but the researchers from the Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT) of the Chinese Academy of Sciences (CAS) determined how to best do this using a microfluidic system and the autofluorescent properties of bacteria.

The results were published in Sensors and Actuators B: Chemical on March 22.

The main functions of this microfluidic platform are organized in a box, which houses the "detection area" the droplets will go through, along with photomultiplier tubes (PMT) and an optical fiber inserted through the chip. A light signal is transmitted to the PMT, and is sorted based on the intensity. There are parameters that can be set both high and low to sort out the target droplets easier.

"A flexible, label-free droplet-based detector allowing bacterial growth phenotype screening may help to expand the scope of rapid bacteria screening in various applications, especially in high-quality industrial breeding," said GE Anle, researcher and first author of the study. 

The researchers determined by using PMT that high densities of bacteria such as E. coli were able to be divided into a sorting channel when exposed to an 800V wave pulse for 5 to 20 milliseconds. Any droplets that did not make it to sorting thresholds were left to fall into the waste channel, and these were typically empty, or negative, droplets. Positive droplets, or droplets with bacteria in them, got deflected into a sorting channel since they were within the given threshold. 

Results from the study indicated sorting efficiency of 95.3% when it comes to cell-containing (positive) droplets, and 91.7% of empty (negative) droplets successfully made it to the waste channel. 

"We anticipate that the mini integrated microfluidic system will serve as a useful platform for further subsequent analyses, including antibiotic resistance, metabolic analysis and industrial strains growth phenotype screening," said DIAO Zhidian, researcher and co-author of the study. 

The microfluidic platform has many advantages compared to the way bacterial growth phenotype screening is done currently. Using an optical fiber to transmit light eliminates the need for a complex lighting system, and most of the core functions are fully integrated into the box structure. 

"The use of determining phenotype using light properties but not relying on fluorescent labels is important, especially because many different cell types may not be compatible with sorting through the use of fluorescent labeling," said corresponding author Prof. MA Bo, from Single-Cell Center of QIBEBT. "The last big advantage is the droplet method: isolating individual bacteria in a droplet can allow for better chances of proliferation, which is of utmost importance in fields involving large-scale industrial breeding of bacteria." 

"We have developed Raman-based flow cytometry tools, such as FlowRACS which improves accuracy, throughput, and stability in profiling dynamic metabolic features of cells, to screen 'high-yield' strains rapidly without the need to label the cell with fluorescence probes. This newly developed microfluidic platform can further screen the 'fast-growing' strains rapidly, so as to achieve the goal of industrial microbial breeding," said Prof. XU Jian, the head of Single-Cell Center of QIBEBT. "Next step, we will further develop the key technologies and equipment platform for single-cell breeding to support the development of industrial biotechnology and synthetic biology."

Source: Chinese Academy of Sciences

Sunday, April 2, 2023

Simplified Screening of Individual Bacterial Species in Biosamples

In medical research and diagnostics, the microbiome, i.e. the microbial colonisation of the intestine, is increasingly gaining attention. A stool sample can be used to precisely analyse the complex microbial ecosystem of the gut. There are basically two methods for this: traditional cultivation on plates with specific culture media or the quite expensive DNA analysis of the stool sample. Both methods on their own are unsatisfactory when it comes to quickly detecting a certain bacterial species in the sample. Researchers from the research training group (RTG) Translational Evolutionary Research (TransEvo) at Kiel University (CAU) have developed a rapid and robust screening method in order to specifically detect and cultivate lactobacilli, bifidobacteria and Bacteroides in stool samples. They published the results in the journal Current Microbiology. "Our method is less expensive and faster than alternative methods usually used to isolate and identify bacteria in complex microbiological samples," explained first author Sofia Borges, a PhD student at the Department of Microbiology and Biotechnology at the Max Rubner-Institut in Kiel. "The method is particularly suitable for screening for certain bacterial species for which there is no exclusive culture medium," added Professor Charles Franz, head of the institute and Associate Professor at the Faculty of Agricultural and Nutritional Sciences at Kiel University. "With this method, we are saving ourselves lengthy procedures for singling out pure bacterial species from potentially non-pure colonies and identifying them."

Bacterial culture is often not selective

Bacterial culturing is the cultivation of microorganisms on a culture medium under controlled conditions, such as temperature. The culture medium that is selected depends on which bacterial species you are looking for in the sample. Selective growth media promote the growth of specific species, while the growth of other species contained in the sample is inhibited. If the species you are looking for is in the sample, it will grow into a colony. That is the ideal scenario. However, the culture media are often not exclusively selective for only one species, but also allow a few other bacterial species to grow.

"In this study, we focused on bifidobacteria, lactobacilli and Bacteroides," said Borges. Bifidobacteria and lactobacilli were chosen because they are relevant to gut health, but are usually not found in large numbers in the intestine. Therefore, even with partially selective media, it is sometimes not possible to cultivate these bacteria. Bacteroides were included in the study as an example of a gram-negative species that is also relevant for gut health and is being further studied by the co-authors of the study.

Detection of pure colonies despite lack of purification

The principle of the method is based on cultivation using three different selective media, DNA extraction, PCR analysis of a specific gene and sequencing. Six stool samples from healthy individuals were used to test this method. Cultivation took place in an anaerobic chamber (without oxygen) for 48 hours at 37 degrees Celsius. Afterwards, well-separated individual colonies were selected for the isolation of the bacteria. The individual colonies were examined according to the study protocol. It was possible to identify the bacterial species contained in all 180 colonies. Most colonies could be assigned to a single species, even though the selective media used not only favoured the growth of the target bacteria but also allowed a few other species to grow. "Some of our colonies contained up to three different species of bacteria. However, we were pleasantly surprised to find that most colonies were pure, despite minimal cultivation and no purification by repeatedly streaking out colonies," explained Borges.

Her working group leader Charles Franz summed up as follows: "Our new method makes it possible to gain insight into the purity of the colonies present on agar plates and to accurately identify the bacteria they contain. It can therefore be useful in providing a speedy, cost-effective and robust overview of bacteria recovered from complex microbiological samples before selecting them for further study."


Borges, A.S.G., Basu, M., Brinks, E. et al. Fast Identification Method for Screening Bacteria from Faecal Samples Using Oxford Nanopore Technologies MinION Sequencing. Curr Microbiol 80, 101 (2023).

Scientists Apply Raman Quantitative 3D Imaging to Microbial Monitoring

Microorganisms are important contributors to the deep-sea sulfur cycle. However, in-situ detection of deep-sea microorganisms faces great challenges due to the extreme complexity of the deep-sea environment, the difficulty of sampling, isolation and cultivation of microorganisms, and the lack of near real-time nondestructive monitoring methods for microbial sulfur metabolism.

To help with this challenge, we can turn to Raman (spectroscopy, not the noodle). 😊

Recently, a research team led by Prof. Zhang Xin and Prof. Sun Chaomin from the Institute of Oceanology of the Chinese Academy of Sciences (IOCAS) achieved long-term, near real-time, non-destructive microbial monitoring through 3D quantitative imaging based on the confocal Raman technology.

The study was published in Microbiology Spectrum

Currently, the process of elemental sulfur production is mainly studied by classical biological and chemical methods, such as X-ray near-edge absorption spectroscopy, high-performance liquid chromatography, transmission electron microscopy, ion chromatography and chemometrics. However, these methods are mainly used to investigate the metabolism of microorganisms at specific time points through sampling, and cannot continuously monitor their metabolic processes on time scales without destroying the cells.

Moreover, some of these methods have complicated sample preparation, which will destroy the in-situ conditions of cells. They may also result in uneven sampling and contamination, making it difficult to achieve continuous in-situ observation.

The confocal Raman 3D imaging is low-cost, rapid, label-free and non-destructive, and has the potential to perfectly combine qualitative, quantitative and visualization.

To demonstrate the potential of this technique, Prof. Zhang's team constructed Raman 3D quantitative in-situ analysis method for microbial communities on solid substrates. It combined optical visualization with Raman quantitative analysis, and could quantitatively characterize the microbial metabolic processes in both temporal and spatial dimensions non-destructively.

The technique has been applied to the in-situ monitoring of the sulfur metabolic processes of the deep-sea cold-seep bacterium E. flavus 21-3. Volume calculations and ratio analysis based on Raman 3D imaging have quantified the growth and metabolism of the microbial colony in different environments. It uncovered the unknown details of microbial growth and metabolism, and provided an important technical support for clarifying the causes of the widely distributed elemental sulfur in deep-sea cold seep.

"To our knowledge, this is the first in-situ nondestructive technique to attempt long-term monitoring of microbial growth and metabolism in solid medium. It supports to rapidly identify metabolites, infer biological pathways, screen the optimal metabolic conditions of microorganisms, and compare the elemental sulfur production rate of different strains," said Prof. Zhang.

"The successful application of this technology not only demonstrates the potential of the method for future visualization and quantitative analysis of microbial processes in situ, but also provides new ideas for studying microorganisms attached to solid surfaces in natural environment," said Prof. Sun.


Wanying He et al, Study of Microbial Sulfur Metabolism in a Near Real-Time Pathway through Confocal Raman Quantitative 3D Imaging, Microbiology Spectrum (2023). DOI: 10.1128/spectrum.03678-22

Tuesday, March 28, 2023

High-Resolution Mass Spectrometric Rapid Identification of Candida auris

A recent study published in the Journal of Fungi used a novel OrbitrapTM high-resolution mass spectrometric technology coupled with liquid chromatography to identify geographically different clades of Candida auris (C. auris) isolates. This proof-of-concept methodology could accurately detect C. auris in the microbiology laboratory.


Over a decade ago, C. auris was first found in East Asia, causing bloodstream infections. Although this fungal infection was initially found in India, South America, South Africa, and the Middle East, it soon prevailed globally. 

C. auris soon became a common nosocomial fungal pathogen, particularly among intensive care unit (ICU) patients. As a result, the Centers for Disease Control and Prevention (CDC) has classified C. auris as an urgent threat pathogen.

An important factor that allows C. auris outbreaks worldwide is the improper identification of yeast pathogens in hospital laboratories. Hence, there is an urgent need for accurate and rapid identification of C. auris in hospital laboratories, which can reduce their transmission in healthcare facilities.

Genomic analysis of worldwide C. auris isolates has indicated that around five clades have emerged in the last 20 years, independently and simultaneously. These five distinct geographically restricted clades are clade I: South Asia, clade II: East Asia, clade III: Africa, clade IV: South America, and clade V: Iran. Each clade differs from the other by around ten thousand single-nucleotide polymorphisms. 

Each clade has differential resistance to antifungal agents; for example, clade I is more resistant to fluconazole, while clade II exhibits susceptibility. Currently, C. auris isolates belonging to these clades have been introduced to many countries worldwide. Scientists have highlighted the importance of quickly identifying and monitoring these clades to restrict further spread. 

C. auris possesses several structurally unique sphingolipids and mannoproteins, enabling it to adhere to medical devices and hospital environments persistently. These proteins also aid in biofilm formation and prevent elimination by common disinfectants.

Several studies have indicated that molecular techniques fail to identify C. auris, whereas matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) technology can accurately identify this fungus at the species level.

The Study and its Findings

102 clinical C. auris strains were selected, representing all five clades. These clades were determined based on a short tandem repeat (STR) typing assay, which was subsequently compared to whole-genome sequencing results.

The current study applied OrbitrapTM high-resolution mass spectrometric technology to identify C. auris based on protein analysis methods. This technique was combined with liquid chromatography (LC) for initial separation. In this method, electrospray ionization (ESI) transfers proteins into the gas phase for ionization and is subsequently introduced to the mass spectrometer (LC-MS).

Mass analysis is conducted by either fragment ions or intact mass (MS) through tandem mass spectrometry (MS/MS). Some of the key features of the OrbitrapTM mass analyzer are a high resolution of up to 200,000, a high mass-to-charge ratio of 6,000, high mass accuracy between 2 and 5 ppm, and a dynamic range greater than 104.

In addition, this method is highly sensitive and can measure the exact mass of a compound. It can also identify minor structural changes due to a translated single nucleotide polymorphism into an amino acid change.

Importantly, this newly developed technology could identify all C. auris isolates with high confidence. Furthermore, it could differentiate C. auris across clades. Even though a limited number of isolates were present from each clade, this spectrometric technology identified C. auris clades with 99.6% identification accuracy.

Based on a principal component analysis (PCA) and a subsequent affinity clustering study, the South Asian, East Asian, and Iranian C. auris clades were more proteomically closely related. Long branches in the affinity clustering analysis suggested that the C. auris strains were present as outliers that required more attention, regardless of the detection technique.

Proteomic typing results indicated the capacity to track strains of the same origin isolated from diverse geographical locations. In the future, more precise matching and alignment of typing schemes (based on next-generation sequencing) is required to build on these results. This would significantly reduce false identifications and classifications of unknown strains associated with new clades or lineage.


Although the workflow linked to mass spectrometry and next-generation sequencing are not directly comparable, their results are similar, i.e., identifying unknown clinical microbes. The standard next-generation sequencing method is a highly time-consuming process that requires many delicate time-intensive quality-control steps, particularly during multiplexed sample runs.

In contrast, the newly developed methodology can provide results within 60 minutes. Therefore, applying the high-resolution OrbitrapTM mass spectrometer to accurately and rapidly identify C. auris clades is an attractive alternative to conventional platforms.

Journal reference:

Jamalian, A. et al. (2023) "Fast and Accurate Identification of Candida auris by High Resolution Mass Spectrometry", Journal of Fungi, 9(2), p. 267. doi: 10.3390/jof9020267,

Source: News-Medical.Net 

Finger-Prick Test Developed for Trichomonas vaginalis

A quick, affordable diagnostic test developed by a Washington State University researcher may help curb one of the most prevalent but least discussed sexually transmitted infections.

More common than chlamydia or gonorrhea, Trichomonas vaginalis, also known as trich, causes no symptoms in about 70% of those infected. Even when asymptomatic, trich is linked to a host of bad health outcomes, including increased susceptibility to HIV, prostate cancer in men and infertility and pregnancy complications in women.

Trich is easily curable with a drug called metronidazole, if diagnosed, and WSU researcher John Alderete has been working for years to improve testing and make it more accessible. His latest development, detailed in the journal Pathogens, is a new finger-prick test that delivers results in five minutes and can be produced for under $20.

“We know a lot about the biology of this organism,” said Alderete, the study lead author and professor in WSU’s School of Molecular Biosciences. “There probably will never be a vaccine for trich simply because the organism is well equipped to evade our immune responses. But I’d argue we don’t need a vaccine. We just need to diagnose people, and once diagnosed, they can be cured.”

Currently trich is often only diagnosed when symptoms are present, which can include genital itchiness and a burning sensation during urination. The tests in use now are focused on diagnosing women and involve a vaginal swab. It takes time to get results, and the tests require trained personnel as well as specialized equipment. Other methods recently approved by the Food and Drug Administration also have similar limitations.

As detailed in this study, the new test requires only a drop of blood to detect an antibody specific to trich. Alderete previously identified this biomarker, an alpha-actinin protein unique to this organism named ACT::SOE3 in earlier research. Both men and women make the antibody when they are infected.

Alderete, working with co-author Hermes Chan from the company MedMira, used the diagnostic platform of the company for his finger-prick test to detect for antibody to the target protein. Similar to COVID‑19 and pregnancy tests, the results are displayed in a window with a dot appearing if the antibody is present, indicating infection. It is a point-of-care diagnostic test meaning positive results lead to immediate treatment and cure of a person. The test does not require specialized training and equipment to administer.

The goal was to meet World Health Organization “ASSURED” standards for disease detection, which stands for affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end users. Since trich is a world-wide problem with an estimated 156 million new cases each year, Alderete hopes the test can ultimately be used in many low-resource countries, particularly in places like Africa where trich is suspected to be a contributing factor in the spread of HIV.  

The test can also have many benefits in the U.S. as well. Alderete estimated there are more than 9.2 million cases annually based on incidence rates and census data. One study found that 50% of pregnant women had persistent undiagnosed infections — a significant concern since trich is associated with pre‑term membrane rupture, preterm birth and low infant birth weight.

The first step is to make more people aware of the problem, Alderete said.

“Trich is the most common STI you’ve probably never heard of,” he said. “This STI may be the most neglected among the other curable STIs. We just have not done a good job in medicine to educate people. One of the major problems is that most people are asymptomatic. In other words, you may have it, but you don’t know you have it until you have a really bad problem.”

Patent protection on the new development is in process.

Tuesday, March 21, 2023

Wayne State Researchers Develop New Technology to Easily Detect Active TB

A team of faculty from Wayne State University has discovered new technology that will quickly and easily detect active Mycobacterium tuberculosis (TB) infection antibodies. Their work, “Discovery of Novel Transketolase Epitopes and the Development of IgG-Based Tuberculosis Serodiagnostics,” was published in a recent edition of Microbiology Spectrum, a journal published by the American Society for Microbiology. The team is led by Lobelia Samavati, M.D., professor in the Center for Molecular Medicine and Genetics in the School of Medicine. Samavati was joined by Jaya Talreja, Ph.D, and Changya Peng, research scientists in Wayne State’s Department of Internal Medicine.

TB remains a global health threat, with 10 million new cases and 1.7 million deaths annually. According to the latest World Health Organization report, TB is the 13th leading cause of death and the second leading infectious killer after COVID-19. Latent tuberculous infection (LTBI) is considered a reservoir for TB bacteria and subjects can progress to active TB. One-third of the world’s population is infected with TB and, on average, 5 to 10% of those infected with LTBI will develop active TB disease over the course of their lives, usually within the first five years after initial infection.

The gold standard tests to determine whether an infection is active TB are the sputum smear and culture tests. However, these methods require collecting sputum, which is time consuming, expensive, requires trained personnel and lacks sensitivity. The current conventional tests  differentiating LTBI from uninfected controls — such as tuberculin skin tests (TST) and/or interferongamma release assay (IGRA) — do not differentiate between active TB infection and latent TB. Despite advances in rapid molecular techniques for TB diagnostics, there is an unmet need for a simple inexpensive point-of-care (POC), rapid non-sputum-based test.

Samavati’s research group has worked for more than 15 years to develop technology for detection of antibodies in various respiratory diseases. Her lab has developed a novel non-sputum based technology and has discovered several novel immune-epitopes that differentialy bind to specific immunoglobulin (IgG) in TB-infected subjects. The levels of epitope-specific IgG  in seum can differentiate active TB from LTBI subjects, healthy contols and other respiratory diseases. This  technology can be used as a simple serum assay non-sputum based serological POC- TB test, which is highly specific and sensitveto diffentiate active TB from LTBI.

“Previously, we developed a T7 phage antigen display platform and after immunoscreening of large sets of serum samples, identified 10 clones that differentially bind to serum antibody (IgG) of active TB patients differentiating TB from other respiratory diseases,” said Samavati. “One of these high-performance clones had homology to the Transketolase (TKT) enzyme of TB bacteria that is an essential enzyme required for the intracellular growth of the bacteria in a host. We hypothesized that abundance of IgG in sera against the identified novel neoantigen that we named as TKTµ may differentiate between active TB, LTBI and other non-TB granulomatous lung diseases such as sarcoidosis. We developed a novel direct Peptide ELISA test to quantify the levels of IgG in serum samples against TKTµ. We designed two additional overlapping M.tb TKT-peptide homologs with potential antigenicity corresponding to M.tb-specific transketolase (M.tb-TKT1 and M.tb-TKT3) and hence standardized three Peptide ELISA (TKTμ, M.tb TKT1 and M.tb TKT3) for the TB serodiagnosis.”

After development and standardization of a direct peptide ELISA for three peptides, the research team tested 292 subjects, and their TKT-peptide ELISA results show that TB patients have significantly higher levels of TKT-specific antibodies compared to patients who were healthy controls and with LTBI. The increased levels of TKT-specific antibodies is presumably associated with growing M.tb bacteria in active TB patients. TKT plays a key role in the switch from the dormancy to proliferative phase and TKT specific IgG may uncover the differences between active TB and LTBI. Thus, IgG-based serodiagnosis of TB with TKT-peptide ELISA is promising.

Currently, commercially available serological TB tests show poor sensitivity and specificity. The ELISA results obtained with the Wayne State team’s discovered TKT peptides yielded high specificity and sensitivity. Their results show that IgG antibodies against transketolase can discriminate active tuberculosis. 

“Our TKT peptide ELISA test requires chemically synthesized TKT peptides to coat the wells in the ELISA plate, less than 100µl blood serum sample from patient, detection reagents and an ELISA plate reader,” said Samavati. “We are extremely enthusiastic about our technology and the fact that with a simple test we can differentiate active TB from LTBI and other respiratory diseases. We believe that our method and TKT peptide ELISA can fit the requirements of the World Health Organization and the Centers for Disease Control and Prevention as a POC screening method.”

The research team has applied a patent application on its technology and is actively seeking companies interested in investing.

This research was supported by the National Heart, Lung and Blood Institute of the National Institutes of Health, grant numbers 113508 and 148089. The Foundation for Innovative New Diagnostics (FIND, Geneva, Switzerland) provided TB and LTBI samples.

The full publication is available at

A New Rapid ‘Glow-in-the-Dark’ Test for Infectious Diseases

PCR testing - the highly sensitive gold standard for Covid-19 detection - requires expensive equipment, expert technicians, and dedicated laboratory facilities, all leading to test times of several hours or even a day. Researchers from Eindhoven University of Technology, Rijnstate Hospital, and Fontys University of Applied Sciences have developed a new test based on a ‘glow-in-the-dark’ approach that could be used to directly detect DNA/RNA of viruses and bacteria with the same sensitivity as a PCR test, and all in just 30 minutes.

“PCR testing is very accurate and was invaluable during the pandemic,” says Maarten Merkx, from the Institute for Complex Molecular Systems (ICMS), department of Biomedical Engineering, and head of the Merkx Lab at TU/e. “But it can take several hours, and often a day, to get the result of a PCR test.”

Part of the time delay with PCR testing, which checks for the presence of viral DNA or RNA in a patient sample, is the need for a thermal cycling process.

“Samples tested via PCR might contain miniscule amounts of viral DNA/RNA. To make this detectable, the sample is subjected to cycles of heating and cooling. This leads to the right conditions for DNA/RNA replication,” says Harm van der Veer, who is a PhD candidate in the Merkx Lab at TU/e. “But expensive equipment is needed, making it unsuitable for use by GPs or other local health facilities, let alone as a home test.”


To address these drawbacks, van der Veer, Merkx and colleagues from TU/e, Rijnstate Hospital, and Fontys University of Applied Sciences have developed a new sensor that can be used at the GP, gives rapid results, and has similar sensitivity and specificity as the PCR test.

And just like the PCR test, their test has a catchy acronym. It’s known as LUNAS. Their work on the test has just been published in the journal ACS Central Science. Van der Veer: “LUNAS stands for luminescent nucleic acid sensor, which means that we have made a bioluminescent sensor to detect the nucleic acids (the molecules that make up DNA or RNA) found in viruses and bacteria.”

The Merkx Lab has considerable experience with bioluminescent sensors. “Many organisms such as fireflies use bioluminescence to produce light. In our sensors, we combine proteins that bind a certain biomolecule (such as an antibody or DNA or RNA) with so-called luciferase enzymes, which emit light upon detection of the target biomolecule in a sample,” says Merkx.  

What’s more, bioluminescent sensors require no external excitation and the ‘glow-in-the-dark’ response can be detected with an ordinary digital camera that you’ll find in a smartphone.

A CRISPR helping hand

When developing LUNAS, the researchers had to solve two major problems: ‘How can the test replicate the thermal cycling amplification of DNA used in PCR testing?’ and ‘How can the sensor accurately bind to the resulting DNA in a rapid manner?’

“Our aim was a test with the sensitivity to detect a single piece of DNA in a microliter sample (a sample that is one millionth of a liter) that also happens to contains lots of other molecules. It’s like trying to find one specific, tiny fish in a sea of quadrillion (10e15) liters of water filled with lots of other fish,” says van der Veer.

First, to quickly produce more viral DNA/RNA copies in a sample, the researchers used so-called recombinase polymerase amplification (RPA), which has been used in other point-of-care (close to the patient) tests in the past, as it works at low, constant temperature unlike PCR.

Second, to ensure specific detection of the DNA/RNA in the sample the researchers turned to CRISPR for help. “Most people associate CRISPR with DNA editing, but at the heart of CRISPR is a very accurate way of identifying and latching onto pieces of DNA”, says van der Veer.

So, the researchers bonded two CRISPR proteins to a split bioluminescent protein (See image). These proteins are designed to recognize and grab pieces of viral DNA copies. When they do so, the two pieces of the bioluminescent protein are joined together and produce blue light. Then, the researchers can detect the blue light using a digital camera.

If there’s no viral DNA/RNA in the sample, little or no blue light is emitted by the sensor. Instead, a reference bioluminescent protein just emits green light.

“However, when viral DNA/RNA is present, within about 10 - 30 minutes there is a marked increase in the blue-to-green light ratio. It’s like a molecular traffic light,” adds Merkx. To validate the accuracy of the LUNAS sensor to detect viral infections, the researchers tested Covid-19 patient samples provided by Rijnstate Hospital.

iGEM beginnings and future directions

LUNAS can be traced all the way back to the iGEM Eindhoven 2019 student project when van der Veer was part of the student team, which worked on making a test for the rapid detection of bacterial pathogens and help fight antimicrobial resistance.

From working on the project, van der Veer developed his interest in biosensors, which led to his masters research, and now PhD research, at the Merkx Lab. “The fruition of my time with iGEM is LUNAS,” says van der Veer.

LUNAS also looks set to be developed to detect other illnesses. “In a collaboration involving Fontys, companies, healthcare professionals, and led by TU/e PhD researcher Yosta de Stigter, we are developing LUNAS to test for sexually transmitted disease (STDs) and a portable microfluidic device to help with its use at point-of-care (near patient or beside) locations,” says Merkx.

Commercialization is also on the minds of van der Veer and Merkx, particularly for healthcare. However, there are key barriers to this adaption in healthcare.

“At the moment, we lack a simple and portable device for the test, which would make it easier for use at GPs or even at home,” notes van der Veer. “In addition, it would be great to have a commercial partner that could produce the tests at a large scale, while also accounting for all of the regulatory steps that need to be adhered to when making such a test.”

Additional information

This research has been supported by the Covid-19 UF fund. The research in this project is also supported by Dutch Research Council (NWO) | Nationaal Regieorgaan Praktijkgericht Onderzoek SIA (NRPO-SIA) project RAAK.PRO02.066.

Paper title: Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation, ACS Central Science, (2023).

Source: Eindhoven University of Technology Newsroom