Wednesday, October 25, 2023

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.


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.

Tuesday, October 24, 2023

Monday, October 23, 2023

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. 

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. 

Monday, October 2, 2023

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.

On-chip pretreatment and rapid AST based directly on positive blood cultures

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."


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.


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.

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.


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


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.