Breathalyzer-Style Test for Instant COVID Results

Scientists at Washington University in St. Louis have developed a breath test that quickly identifies those who are infected with the virus that causes COVID-19. The device requires only one or two breaths and provides results in less than a minute.

The study is available online in the journal ACS Sensors. The same group of researchers recently published a paper in the journal Nature Communications about an air monitor they had built to detect airborne SARS-CoV-2 — the virus that causes COVID-19 — within about five minutes in hospitals, schools and other public places.

The new study is about a breath test that could become a tool for use in doctors’ offices to quickly diagnose people infected with the virus. If and when new strains of COVID-19 or other airborne pathogenic diseases arise, such devices also could be used to screen people at public events. The researchers said the breath test also has the potential to help prevent outbreaks in situations where many people live or interact in close quarters — for example aboard ships, in nursing homes, in residence halls at colleges and universities, or on military bases.

“With this test, there are no nasal swabs and no waiting 15 minutes for results, as with home tests,” said co-corresponding author Rajan K. Chakrabarty, PhD, the Harold D. Jolley Career Development Associate Professor of Energy, Environment & Chemical Engineering at the McKelvey School of Engineering. “A person simply blows into a tube in the device, and an electrochemical biosensor detects whether the virus is there. Results are available in about a minute.”

Technology Behind the Test

The biosensor used in the device was adapted from an Alzheimer’s disease-related technology developed by scientists at Washington University School of Medicine in St. Louis to detect amyloid beta and other Alzheimer’s disease-related proteins in the brains of mice. The School of Medicine’s John R. Cirrito, PhD, a professor of neurology, and Carla M. Yuede, PhD, an associate professor of psychiatry — both also co-corresponding authors on the study — used a nanobody, an antibody from llamas, to detect the virus that causes COVID-19.

Chakrabarty and Cirrito said the breath test could be modified to simultaneously detect other viruses, including influenza and respiratory syncytial virus (RSV). They also believe they can develop a biodetector for any newly emerging pathogen within two weeks of receiving samples of it.

“It’s a bit like a breathalyzer test that an impaired driver might be given,” Cirrito said. “And, for example, if people are in line to enter a hospital, a sports arena or the White House Situation Room, 15-minute nasal swab tests aren’t practical, and PCR tests take even longer. Plus, home tests are about 60% to 70% accurate, and they produce a lot of false negatives. This device will have diagnostic accuracy.”

Development Journey

The researchers began working on the breath test device — made with 3D printers — after receiving a grant from the National Institutes of Health (NIH) in August 2020, during the first year of the pandemic. Since receiving the grant, they’ve tested prototypes in the laboratory and in the Washington University Infectious Diseases Clinical Research Unit. The team continues to test the device, to further improve its efficacy at detecting the virus in people.

For the study, the research team tested COVID-positive individuals, each of whom exhaled into the device two, four, or eight times. The breath test produced no false negatives and gave accurate reads after two breaths from each person tested. The clinical study is ongoing to test COVID-positive and -negative individuals to further test and optimize the device.

Strain Detection and Operation

The researchers also found that the breath test successfully detected several different strains of SARS-CoV-2, including the original strain and the omicron variant, and their clinical studies are measuring active strains in the St. Louis area.

To conduct the breath test, the researchers insert a straw into the device. A patient blows into the straw, and then aerosols from the person’s breath collect on a biosensor inside the device. The device then is plugged into a small machine that reads signals from the biosensor, and in less than a minute, the machine reveals a positive or negative finding of COVID-19.

Future Prospects

Clinical studies are continuing, and the researchers soon plan to employ the device in clinics beyond Washington University’s Infectious Diseases Clinical Research Unit. In addition, Y2X Life Sciences, a New York-based company, has an exclusive option to license the technology. That company has consulted with the research team from the beginning of the project and during the device’s design stages to facilitate possible commercialization of the test in the future.

Reference

“Rapid Direct Detection of SARS-CoV-2 Aerosols in Exhaled Breath at the Point of Care” by Dishit P. Ghumra, Nishit Shetty, Kevin R. McBrearty, Joseph V. Puthussery, Benjamin J. Sumlin, Woodrow D. Gardiner, Brookelyn M. Doherty, Jordan P. Magrecki, David L. Brody, Thomas J. Esparza, Jane A. O’Halloran, Rachel M. Presti, Traci L. Bricker, Adrianus C. M. Boon, Carla M. Yuede, John R. Cirrito and Rajan K. Chakrabarty, 27 July 2023, ACS Sensors. DOI: 10.1021/acssensors.3c00512

The study was funded by the National Institutes of Health (NIH) RADx-Rad program. Grant numbers U01 AA029331 and U01 AA029331-S1. Additional funding from the National Institute of Neurological Disorders and Stroke Intramural Research Program, the Uniformed Services University of Health Sciences, and the NIH SARS-CoV-2 Assessment of Viral Evolution (SAVE) Program.

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Regulatory Acceptance of Rapid Methods: A New Video Tutorial

Want to learn more about rapid microbiological methods (RMM) and their acceptance by worldwide regulatory authorities? Click on the following link to visit my RMM Regulatory Page to watch this 12-minute tutorial.

http://rapidmicromethods.com/files/regulatory.php. 

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A New Portable DNA Sensor to Detect Viral and Bacterial Pathogens in Wastewater

Scientists from the Indian Institute of Technology Bombay (IIT Bombay) have developed a low- cost, portable device designed to detect DNA in wastewater and other water bodies to aid in the early detection of viral and bacterial pathogens. The sensor was shown to be able to detect the presence of pathogens, such as E. coli bacteria and bacteriophage phi6 virus, in sewage and water bodies.

Wastewater surveillance involves monitoring wastewater and sewage water in an area for pathogens to ascertain the health of a community. Studies have shown that the concentration of pathogens in wastewater can be used as an accurate measure for the population-level spread of a disease. “The origin of monitoring wastewater and sewage for pathogen detection and outbreak of epidemics goes all the way back to 1939 when the initial application of wastewater surveillance for detecting poliovirus on a community level was demonstrated,” says Prof. Siddharth Tallur, from the Department of Electrical Engineering, IIT Bombay and a part of the team that developed the new portable sensor.

In recent times, the COVID-19 pandemic has once again brought out the importance of wastewater surveillance. Some of those infected with the SARS-CoV-2 were asymptomatic, showing no external signs of infection, and hence posing challenges to track them with clinical surveillance alone. Data from wastewater surveillance complemented clinical surveillance data by providing valuable estimates as to how many individuals were infected and which SARS-CoV-2 variants were circulating in a community. “Wastewater-based epidemiology serves as a tool for data collection from populations which lack adequate access to healthcare and large-scale individual- level diagnostic testing,” adds Prof. Tallur.

Currently, the most commonly used method for detecting disease-causing agents is the real-time quantitative polymerase chain reaction (RT-qPCR), a technique known for its high specificity and sensitivity. However, the qPCR method requires expensive probes and trained personnel to administer, thereby limiting its application to well-equipped laboratories.

The COVID-19 pandemic saw the invention of several new biosensors and detectors that could pick out the SARS-CoV-2 virus from wastewater samples. There have also been smartphone-based sensors that detect changes in colour in a sample denoting the presence of pathogen DNA. These, however, either sacrifice sensitivity or require expensive reagents and equipment, sterile lab conditions and experts to operate. The new portable sensor developed at IIT Bombay significantly reduces these limitations. It is highly sensitive to any DNA present in a sample, yet keeps the costs low.

The IIT Bombay device functions by detecting colour changes in samples created by the interaction of DNA with methylene blue (MB) dye. Intercalation is the process by which molecules, such as methylene blue, insert themselves in between bases of DNA. This causes a change in the property of the material to absorb light of different wavelengths, thereby causing a change in its colour. In a sample prepared for testing, this leads to a change in the colour of the sample. The colourimetric sensor system is designed around an indigenously built circuit, called a phase-sensitive detection circuit, which detects this change in colour. The sensor consists of a sample holder connected to the colourimetric sensor. Once a sample is placed in the sample holder, the sensor picks up any colour change in the sample due to DNA, which is then converted to a voltage signal for measurement and recording. The IIT Bombay team has also developed a mobile application that can read this voltage signal via Bluetooth and display the information on a smartphone.

PCR is a process used to multiply a specific DNA segment. Unpurified PCR products, along with the DNA, contain other organic materials like enzymes and nucleotides along with chemical contaminants, like primers and buffers used for the process. The sensor proved to be capable of detecting DNA in unpurified PCR products and distinguishing these from control samples, containing purified DNA.

The phase-sensitive detection circuit, which was completely designed and built at IIT Bombay, is constructed from low-cost semiconductor integrated circuit components and a low-power LED light source. Methylene blue is a widely used dye – easy to obtain and inexpensive. These factors have allowed the researchers to keep the cost of manufacturing and operating the sensor low. “The technology developed in our work holds promise for the realisation of a truly cost-effective solution for wastewater-based epidemiology,” opines Prof. Tallur.

The sensor is, however, not without its limitations. The use of methylene blue dye means reduced specificity of the device. According to Prof. Tallur “It (methylene blue) will bind with any DNA present in the sample to which it is added, and therefore the overall specificity of the sensor is determined by purity and choice of the primers used for target amplification in PCR (chemicals added to target DNA of a specific pathogen).” The research team envisions that with advancements like other dyes with higher specificity, target-specific probes and robust microfluidic chips, the system can be enhanced for improved sensitivity, specificity, and robustness.

The development of the device is in its nascent stages and with time more improvements are expected. “We are working on developing more time-efficient and low-cost methods for sample pre-processing, and robust and highly specific assays that can be used for optical and electrochemical DNA sensors. We have filed some patent applications based on these ideas and work, and more will be filed in future as we continue to make progress in this direction,” remarks Prof. Tallur about the future of the new device.

The successful application of this sensor could prove crucial for regular surveillance and early warning systems for potential epidemic outbreaks. It has the potential to revolutionise environmental screening methods for viral and bacterial infections, enabling early detection and prevention strategies to be initiated at the outset. The most important aspect is that it is possible to achieve all of this without a significant dent in the finances of institutions and nations.

Reference: Portable absorbance platform for sensing of viral and bacterial nucleic acid leveraging intercalation with methylene blue: Application for wastewater-based epidemiology. Biosensors and Bioelectronics: X Volume 14, September 2023. 

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Lateral Flow test for Rapidly Detecting Gingivitis Bacteria

Researchers at the University of Cincinnati have developed a lateral flow assay that can detect bacterial toxins from Porphyromonas gingivalis, the causative bacteria for gingivitis. The technology could make it easier and faster to identify early-stage gingivitis, which can lead to periodontitis and eventual tooth loss, as well as contributing to a variety of other diseases such as stroke and heart disease. The lateral flow assay requires a small saliva sample, and can provide results very quickly, but does require the saliva sample to be pre-treated with potato starch to deactivate salivary amylase, an enzyme that can interfere with the assay.

The humble lateral flow assay grew in prominence during the COVID-19 pandemic as a quick at-home method to check your COVID status, but this technology was already a staple of such applications as pregnancy testing. Now, researchers are increasingly aware of its utility as a rapid point-of-care diagnostic technology and are beginning to apply it to the detection of other diseases. In this instance, these researchers at the University of Cincinnati have developed a lateral flow assay to detect the bacteria responsible for gingivitis.

Gingivitis is caused by P. gingivalis, which typically starts as mild gum inflammation. However, this can spread to other parts of the periodontal tissue, causing damage to soft tissue and bone that stabilize our teeth. This damage can eventually lead to tooth loss. Moreover, researchers have also linked P. gingivalis to other conditions, including cardiovascular diseases, rheumatoid arthritis, and even neurodegenerative diseases such as Alzheimer’s disease.  

There are lab-based tests available to detect P. gingivalis, but compared with a lateral flow test, they are complex, slow, expensive, and lack portability. If a diagnostic technique is too expensive, time consuming and inconvenient, then patients or clinicians will only tend to seek it out or recommend it if symptoms have already developed. However, for routine testing and health screening, a convenient, rapid, and point-of-care test is much preferred. A lateral flow test for gingivitis, for example, could be administered by a dentist every time someone undergoes a routine dental checkup.   

The assay detects a bacterial endotoxin released into the saliva by P. gingivalis through a simple immunoassay, whereby antibodies capture and identify the toxin. An enzyme present in saliva called amylase can interfere with this, so the assay requires the saliva to be pretreated with potato starch to deactivate this enzyme. In the future, you may be able to use such lateral flow assays to conveniently detect a wide variety of pathogens and biomarkers, and you can thank SARS-CoV-2 for the privilege. 

Reference: Salivary endotoxin detection using combined mono/polyclonal antibody-based sandwich-type lateral flow immunoassay device. Sens. Diagn., 2023, Advance Article. 

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Microfluidic Chip for Rapid Antimicrobial Susceptibility Testing Directly from Positive Blood Cultures

Viable bacteria in the blood, i.e., bacteremia, can lead to bloodstream infection (BSI) and sepsis, a syndromic, often fatal, inflammatory response.

Rapid and accurate antimicrobial prescriptions are critical to decreasing mortality in BSI patients. However, traditional antimicrobial susceptibility testing (AST) for BSI is time-consuming and tedious, leading clinicians to rely primarily on their experience when prescribing treatment.

Responding to the need for faster diagnostic tools, researchers from Shandong University, the Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT) of the Chinese Academy of Sciences (CAS), and the Affiliated Hospital of Qingdao University have developed an integrated microfluidic chip (BSI-AST chip) for rapid AST from positive blood cultures (PBCs). Using the chip, the process from bacteria extraction to getting AST results takes less than 3.5 hours, thus promising to be a powerful new tool in managing bloodstream infections.

The study was published in Analytical Chemistry.

"Traditional AST methods currently require at least two days to yield results following a positive blood culture. The delay in diagnosis compels the administration of empirical antibiotics, risking the aggravation of the patient's condition and fostering the emergence of antibiotic resistance," said Prof. MA Bo from the Single-Cell Center at QIBEBT, co-author of the study. "Therefore, there is an urgent need for new technologies that can provide accurate and timely diagnostics and drug susceptibility testing."

In this study, the researchers designed a BSI-AST chip capable of extracting bacteria directly from PBCs within 10 minutes—providing rapid AST results requires an additional three hours.

In a proof-of-concept study, the BSI-AST chip demonstrated its effectiveness by conducting direct AST on artificial PBCs containing E. coli, testing against 18 antibiotics, with results in less than 3.5 hours.

Moreover, the integrated chip was applied to the diagnosis of clinical PBCs, showing a categorical agreement of 93.3% with standard clinical methods. The reliable and rapid AST results of the chip highlight its great potential in clinical diagnosis.

"In previous studies, microfluidic devices were mainly designed for purification and concentration of viable microorganisms derived from subculture or urine samples with simple composition," said ZHU Meijia, a doctoral student from Shandong University and first author of the study. "The practical utilization of these devices faced significant challenges due to the absence of on-chip complex sample preparation processes."

XU Teng, assistant research fellow and contributing author from the Single-Cell Center at QIBEBT, said that the BSI-AST chip was a "significant advancement" since it could work directly from PBCs without the need for a subculture.

The researchers achieved rapid extraction and enrichment of bacteria from PBCs by introducing a separator gel to the microfluidic chip for the first time. Centrifugal microfluidic enrichment technology also was central to the process. Furthermore, the chip's multiplexing analysis capability through antibiotic drying and array parallelization support clinicians in optimizing antibiotic therapy for BSI patients.

The BSI-AST chip also provides a rapid and convenient solution for sample pretreatment when combined with Clinical Antimicrobial Susceptibility Test Ramanometry (CAST-R), an instrument that the team invented, according to Prof. XU Jian, the head of the Single-Cell Center at QIBEBT.

"Rapid AST in blood culture is significant for patients with clinical sepsis and has the potential to save lives," said Prof. CHENG Yongqiang of Shandong University, the study's corresponding author. Prof. CHENG also noted the role of such technology in "combating the serious threat of microbial resistance to humanity."

Reference

Integrated Microfluidic Chip for Rapid Antimicrobial Susceptibility Testing Directly from Positive Blood Cultures. Meijia Zhu, Teng Xu, Yongqiang Cheng, et al. Anal. Chem. 2023, 95, 38, 14375–14383. doi.org/10.1021/acs.analchem.3c02737. 

Abstract

Rapid and accurate antimicrobial prescriptions are critical for bloodstream infection (BSI) patients, as they can guide drug use and decrease mortality significantly. The traditional antimicrobial susceptibility testing (AST) for BSI is time-consuming and tedious, taking 2–3 days. Avoiding lengthy monoclonal cultures and shortening the drug sensitivity incubation time are keys to accelerating the AST. Here, we introduced a bacteria separation integrated AST (BSI-AST) chip, which could extract bacteria directly from positive blood cultures (PBCs) within 10 min and quickly give susceptibility information within 3 h. The integrated chip includes a bacteria separation chamber, multiple AST chambers, and connection channels. The separator gel was first preloaded into the bacteria separation chamber, enabling the swift separation of bacteria cells from PBCs through on-chip centrifugation. Then, the bacteria suspension was distributed in the AST chambers with preloaded antibiotics through a quick vacuum-assisted aliquoting strategy. Through centrifuge-assisted on-chip enrichment, detectable growth of the phenotype under different antibiotics could be easily observed in the taper tips of AST chambers within a few hours. As a proof of concept, direct AST from artificial PBCs with Escherichia coli against 18 antibiotics was performed on the BSI-AST chip, and the whole process from bacteria extraction to AST result output was less than 3.5 h. Moreover, the integrated chip was successfully applied to the diagnosis of clinical PBCs, showing 93.3% categorical agreement with clinical standard methods. The reliable and fast pathogen characterization of the integrated chip suggested its great potential application in clinical diagnosis.

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Rapid and Point-of-Care Eye Test Detects Aspergillus Keratitis

Point-of-care diagnostics similar to home COVID-19 tests were able to detect microbial keratitis (MK) caused by Aspergillus fungus with good accuracy, according to a prospective study.

An Aspergillus-specific lateral-flow device already FDA-approved for pulmonary aspergillosis testing was able to detected MK with high sensitivity for both corneal scrape (0.89, 95% CI 0.74-0.95) and swab samples (0.94, 95% CI, 0.73-1.00).

Overall accuracy was 0.94 (95% CI 0.90-0.97) and 0.88 (95% CI 0.73-0.96), respectively, reported Bethany Mills, PhD, of the University of Edinburgh, Scotland, and colleagues in JAMA Ophthalmology.

The study was "a useful proof-of-concept" for rapid detection of pathogens causing the infection, which could allow a more targeted treatment approach, Mills told MedPage Today.

Microbes causing the condition are bacteria in half of cases and fungi like Aspergillus in the other half, but clinicians usually can't distinguish without testing, Mills said. That makes microbiological diagnosis especially important, she said, but it requires highly skilled lab workers at tertiary care centers. These facilities can be hundreds of kilometers from sites in Africa and Asia, she said, and testing can take a week.

The disease is more severe and more common in tropical and subtropical regions, where it often causes blindness and eye loss, particularly in poorer nations.

The disease "is an acute ophthalmic emergency," Mills said. "Across Asia and Africa, it is the second most common cause of single-eye vision loss following cataracts. In these regions, 60% of patients are left with moderate or worse visual outcomes, and up to 15% require surgery that's often unsuccessful. Current treatment strategies include topical antimicrobials, antibiotics, and/or antifungals, and then surgery."

For the study, researchers used a lateral-flow device known as AspLFD that is FDA-approved to test for pulmonary aspergillosis and costs about $12 in Western countries. "It has never been looked at in the context of keratitis," Mills said. "To our knowledge, no lateral flow or point-of-care device has been looked at for keratitis."

As Mills explained, "think of this as being like a COVID lateral flow test, which everyone is familiar with. Instead of swabbing your nose, your cornea is scraped or swabbed to retrieve the sample, with a numbing drop added first. The sample is then placed into a buffer and then put onto the lateral flow test."

The researchers suggested "swabs could be used to collect corneal samples from patients with suspected MK in settings where the routine method of specimen collection by scraping is not possible, such as primary care."

Their group tested the diagnostic on corneal swab and scrape samples taken from 198 individuals ages 15 or older with MK (63.6% male, mean age 51) from 2022-2023 at a single eye hospital in Madurai, India. Some had already been treated with antibiotics, antifungals, or both at the time of sampling.

The samples were placed into the AspLFD devices, and a laboratory microbiologist inspected them after 20 minutes.

Among corneal scrape samples, 39 were positive for Aspergillus according to the AspLFD test. A reference culture test revealed that 31 were actually positive and eight were false positives. Of the 159 negative scrape samples by the AspLFD test, the reference culture test revealed that 155 were negative and four were false negatives.

Twenty corneal swab samples were positive per the AspLFD test, of which reference culture test revealed that 16 were actually positive and four were false positives. Another 20 samples were deemed negative by the test, with one being a false negative by the reference culture test.

The researchers noted that "while not included within the formal AspLFD analysis, 5 of the 76 culture-negative, fungal smear-positive scrape samples also had positive AspLFD results, suggesting that these patients likely had a missed Aspergillus species infection."

The study authors noted limitations. For one, the subjects often had "relatively advanced" stages of MK, making it unclear how the test would work in those with less severe cases. Researchers also had to eyeball the test results looking for the band to determine whether the results were positive or negative. Some of the bands were faint. To improve resolution, the researchers looked at photos of the devices that were taken using open-source software and a smartphone.

What's next? While testing kits may not be readily available, "we believe that ophthalmologists may start using them now," said study co-author Venkatesh Prajna, MD, of Aravind Eye Hospital in India, in an interview.

Reference:

Rapid Point-of-Care Identification of Aspergillus Species in Microbial Keratitis. Rameshkumar Gunasekaran, MSc; Abinaya Chandrasekaran, MS; Karpagam Rajarathinam, CMLT; et al. JAMA Ophthalmol. Published online September 28, 2023. doi:10.1001/jamaophthalmol.2023.4214

Abstract

Importance:  Microbial keratitis (MK) is a common cause of unilateral visual impairment, blindness, and eye loss in low-income and middle-income countries. There is an urgent need to develop and implement rapid and simple point-of-care diagnostics for MK to increase the likelihood of good outcomes.

Objective:  To evaluate the diagnostic performance of the Aspergillus-specific lateral-flow device (AspLFD) to identify Aspergillus species causing MK in corneal scrape and corneal swab samples of patients presenting with microbial keratitis.

Design, Setting, and Participants:  This diagnostic study was conducted between May 2022 and January 2023 at the corneal clinic of Aravind Eye Hospital in Madurai, Tamil Nadu, India. All study participants were recruited during their first presentation to the clinic. Patients aged 15 years or older met the eligibility criteria if they were attending their first appointment, had a corneal ulcer that was suggestive of a bacterial or fungal infection, and were about to undergo diagnostic scrape and culture.

Main Outcomes and Measures:  Sensitivity and specificity of the AspLFD with corneal samples collected from patients with MK. During routine diagnostic scraping, a minimally invasive corneal swab and an additional corneal scrape were collected and transferred to aliquots of sample buffer and analyzed by lateral-flow device (LFD) if the patient met the inclusion criteria. Photographs of devices were taken with a smartphone and analyzed using a ratiometric approach, which was developed for this study. The AspLFD results were compared with culture reports.

Results:  The 198 participants who met the inclusion criteria had a mean (range) age of 51 (15-85) years and included 126 males (63.6%). Overall, 35 of 198 participants with corneal scrape (17.7%) and 17 of 40 participants with swab samples (42.5%) had positive culture results for Aspergillus species. Ratiometric analysis results for the scrape samples found that the AspLFD achieved high sensitivity (0.89; 95% CI, 0.74-0.95), high negative predictive value (0.97; 95% CI, 0.94-0.99), low negative likelihood ratio (0.12; 95% CI, 0.05-0.30), and an accuracy of 0.94 (95% CI, 0.90-0.97). Ratiometric analysis results for the swab samples showed that the AspLFD had high sensitivity (0.94; 95% CI, 0.73-1.00), high negative predictive value (0.95; 95% CI, 0.76-1.00), low negative likelihood ratio (0.07; 95% CI, 0.01-0.48), and an accuracy of 0.88 (95% CI, 0.73-0.96).

Conclusions and Relevance:  Results of this diagnostic study suggest that AspLFD along with the ratiometric analysis of LFDs developed for this study has high diagnostic accuracy in identifying Aspergillus species from corneal scrapes and swabs. This technology is an important step toward the provision of point-of-care diagnostics for MK and could inform the clinical management strategy.

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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.

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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

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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.

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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

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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).

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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. 

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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 

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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. 

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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.

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Biochip Detects Multiple Viruses, Cancers, or Toxins in Minutes

Rapid COVID-19 tests gave many people a firsthand appreciation for the value of quick and cheap diagnostics. Now, researchers have shown how to conduct thousands of rapid molecular screenings simultaneously, using light to identify target molecules snared on top of an array of tiny silicon blocks. In theory, the tool could be used to spot 160,000 different molecules in a single square centimeter of space. Developed to spot gene fragments from the SARS-CoV-2 virus and other infectious organisms, the technology should also be able to identify protein markers of cancer and small molecules flagging toxic threats in the environment.

“This technology could have a big role to play in how we detect things in the environment,” says Chris Scholin, a molecular biologist and president and CEO of the Monterey Bay Aquarium Research Institute. The tool could also be useful in clinical diagnostics, he adds, although it has several competing technologies already in wide use.

Genetic tests are nothing new. Most of these technologies rely on measuring light absorption or emission from probe molecules tailored to latch onto the target gene. But to produce a signal large enough to detect, most of the technologies rely on amplifying techniques such as polymerase chain reaction to produce many copies of the target before trying to detect them, adding to the cost and time of the tests.

Researchers have devised a variety of more sensitive technologies. “But previous sensors have not been able to detect a wide range of target molecules,” from very low to very high abundance, says Jennifer Dionne, an applied physicist at Stanford University.

In hopes of getting around these problems, Dionne and her colleagues turned to an optical detection approach that relies on metasurfaces, arrays of tiny silicon boxes—each roughly 500 nanometers high, 600 nanometers long, and 160 nanometers wide—that focus near-infrared light on their top surface. This focusing makes it easy for a simple optical microscope to detect the shift in the wavelength of light coming from each silicon block, which varies depending on what molecules sit on top.

A rapid screen detects gene fragments tethered to arrays of silicon boxes, each just 500 nanometers tall and 600 nanometers across.

To test the idea, the researchers tethered single-stranded gene fragments 22 nucleotides long to the silicon boxes and immersed the array in a buffer solution. When they added the complementary DNA strands to the solution, the strands quickly bound to the tethered ones, shifting the wavelength of light emitted from the surface of each box. Dionne and her colleagues report that their setup could detect the presence of as few as 4000 copies of target genes per microliter, a result they published in Nature Communications.

That’s a concentration typically present in a nasal sample from a person infected with SARS-CoV-2. So the technique could allow doctors to detect viral infections without first having to amplify the genetic material from a patient, Dionne says. Perhaps as important, she notes, an array can be designed to reveal how much target DNA has bound, making it possible to detect in minutes not just whether a particular virus is present, but how intense the infection is. Such information could help doctors tailor their treatments. Current tests can also do this, but they normally take several hours to amplify the genetic material and quantify the results.

Scholin argues that the technology could find more immediate widespread use in tracking molecules outside the lab or doctor’s office. For example, environmental scientists currently use genetic probes to detect toxic algae in waterways. But this normally requires added processing steps to amplify target genes and then test for their abundance, which can take hours, if not days, of lab work.

In that situation, the new technique’s speed could be a game changer, Scholin says. Another enticing option, he says, is to tether antibodies on top of the silicon boxes. This might allow researchers to directly grab the corresponding antigen, whether a toxin or a protein marker of disease. He hopes to use the Stanford team’s detectors to see whether they can detect microbial toxins in the water directly on the fly. “That would have a real impact on people, ecology, and wildlife,” he says.

Dionne and her colleagues have formed a company called Pumpkinseed Bio to commercialize their new detectors, specifically aimed at detecting minute levels of proteins and other molecules that can’t readily be amplified to make them easier to detect. And because only a small number of silicon blocks would be needed to spot individual target molecules, researchers should be able to craft arrays to track a multitude of disease biomarkers simultaneously. “We hope to look at many disease states at the same time,” says Jack Hu, a former graduate student in Dionne’s lab and head of the new startup. “That’s the vision.”

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Dogs May Be More Sensitive at Detecting COVID-19 More Rapidly and Accurately than Current Tests

Our fur babies may provide a cheaper, faster and more effective way to detect COVID-19, and could be a key tool in future pandemics, a new review of recent research suggests. The review, published in De Gruyter’s Journal of Osteopathic Medicine, found that scent dogs are as effective, or even more effective, than conventional COVID-19 tests such as RT-PCR.

Dogs possess up to 300 million olfactory cells, compared to just 5 or 6 million in humans, and use one-third of their brains to process scent information, compared with just 5% for humans. Dogs trained to recognize specific volatile organic compounds created in the body during disease have successfully identified patients with certain cancers, Parkinson’s and diabetes.

Prof. Tommy Dickey of the University of California, Santa Barbara and Heather Junqueira of BioScent Detection Dogs reviewed 29 studies where dogs were used to detect COVID-19. The studies were performed using over 31,000 samples by over 400 scientists from more than 30 countries using 19 different dog breeds. In some studies, the scent dogs sniffed people directly, sometimes in public places as a health screening. In others, the dogs sniffed patient samples such as sweat, saliva or urine samples.

In the majority of studies, the scent dogs demonstrated similar or better sensitivity and specificity than the current gold-standard RT-PCR tests or antigen tests. In one study, four of the dogs could detect the equivalent of less than 2.6 x 10−12 copies of viral RNA per milliliter. This is equivalent to detecting one drop of any odorous substance dissolved in ten and a half Olympic-sized swimming pools and is three orders of magnitude better than modern scientific instruments. 

The dogs could detect COVID-19 in symptomatic, pre-symptomatic and asymptomatic patients, along with new COVID variants and even long COVID. A major benefit of using the dogs was their speed – they could provide a result in seconds to minutes, and did not require expensive lab equipment or create mountains of plastic waste, unlike conventional diagnostic approaches.

“Although many people have heard about the exceptional abilities of dogs to help humans, their value to the medical field has been considered fascinating, but not ready for real-world medical use,” said Prof. Dickey. “Having conducted this review, we believe that scent dogs deserve their place as a serious diagnostic methodology that could be particularly useful during pandemics, potentially as part of rapid health screenings in public spaces. We are confident that scent dogs will be useful in detecting a wide variety of diseases in the future."

Prof. Dickey and Heather Junqueira added that they feel that the impressive international COVID scent dog research described in their paper, perhaps for the first time, demonstrates that medical scent dogs are ready for mainstream medical applications.

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Blood culture vs. Metagenomic Next-Generation Sequencing for the Detection of Pathogenic Microbes in Patients with Bloodstream Infections

A recent study published in Scientific Reports compared the detection of pathogenic microbes between blood culture and metagenomic next-generation sequencing (mNGS) in patients suspected of bloodstream infections (BSIs).

Background

BSI can manifest as fungemia, viremia, and bacteremia, increasing hospitalization duration and costs. BSI incidence has increased over the past years. As such, research focus on the early identification of pathogens has been increasing. mNGS offers several advantages over a conventional blood culture assay, such as high speed and a wide range of pathogen detection. Nevertheless, mNGS is not commonly used for BSIs due to its high costs.

About the study

In the present study, researchers compared the pathogen detection consistency of blood culture assay and mNGS. They retrospectively evaluated patients with suspected BSIs admitted to the emergency department of a Chinese hospital between January 2020 and June 2022. Eligible patients aged 16 or older had chills and body temperature above 38.5 °C, with antibiotic usage longer than three days. mNGS was performed on the day of sampling.

Blood samples were collected from two anatomical sites and cultured for up to seven days using standard microbiological procedures. For mNGS, DNA was isolated, and DNA libraries were prepared. Quality control-passed libraries were sequenced. Short, adapter, low-complexity, and low-quality reads were removed. The remaining reads were aligned to microbial genome databases.

Unique reads had > 90% identity and > 80% alignment, with the ratio of sub-optimal to optimal alignment score lower than 0.8. Patients’ medical records were reviewed. The team obtained data on demographics, comorbidities, laboratory tests, mechanical ventilation, central venous intubation, sequential organ failure assessment (SOFA), and in-hospital death. Logistic regression was performed to identify risk factors for a positive blood culture or mNGS.

Findings

The study included 99 patients suspected of BSIs. They were predominantly males and aged 63 on average. Inflammatory indicators, such as C-reactive protein (CRP), white blood cells (WBCs), and procalcitonin (PCT), were elevated in patients. Mechanical ventilation and central venous intubation were required for 36.3% and 56.5% of patients, respectively.

The median SOFA score was six. In-hospital death occurred in 37 patients. There was a statistically significant difference in the number of patients positive on a blood culture assay and mNGS. Sixty-five patients were positive on mNGS compared to 12 on blood culture assays. mNGS detected a virus in 22 patients and fungi or bacteria in the remaining patients.

By contrast, blood cultures only detected fungi or bacteria. The most common pathogens detected through blood cultures were Staphylococcus haemolyticus, Klebsiella pneumoniae, and Enterococcus faecalis. Escherichia coli, K pneumoniae, and Salmonella enterica were the most commonly identified pathogens in mNGS.

The detection rate was significantly higher with mNGS than with blood culture; the concordance in detecting fungi and bacteria was 12% between mNGS and blood culture. Logistic regression identified lower WBC count, body mass index (BMI), and elevated CRP as risk factors for pathogen detection in mNGS.

Increased age and CRP, rheumatic diseases, and alcohol abuse were the risk factors for detecting fungi or bacteria in mNGS. Current smoking status and gender were identified as the risk factors for positive blood cultures. In-hospital mortality rates were 38.4% and 35.2% in mNGS-positive and -negative cases and 38.4% and 37.2% in blood culture-positive and -negative cases, respectively.

Conclusions

Early diagnosis of BSIs is crucial; blood cultures are currently the gold standard for detection, producing results within three to five days. Besides, other techniques based on multiplex real-time polymerase chain reaction (PCR) and metagenomics have been increasing. Of these, mNGS offers rapid results and could help improve patient management.

The rate of positive blood cultures was 13.1%, consistent with previous reports. While mNGS and blood cultures identified fungi and bacteria, only mNGS detected viruses. The positive rate of mNGS was 3.31-fold higher than that of blood cultures. The mortality rate was 38.4%, higher than in prior studies.

There were no significant differences in mortality rates between culture- or mNGS-positive and negative patients. The study revealed higher positivity rates with mNGS than with conventional blood cultures, and their combined use could maximize the detection rate of bloodstream pathogens.

Reference

Zhou, Y. et al. (2023) "Comparison of pathogen detection consistency between metagenomic next-generation sequencing and blood culture in patients with suspected bloodstream infection", Scientific Reports, 13(1). doi: 10.1038/s41598-023-36681-5 https://www.nature.com/articles/s41598-023-36681-5

Source: News-Medical.Net

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