Friday, February 17, 2023

Rapid C. difficile Testing and Diagnosis Improves Patient Outcomes and Reduces Cost

When it comes to infection diagnosis and treatment, the sooner the better.

Implementing a rapid near patient testing program around suspected Clostridiodes difficile infections (CDI) cases in hospital settings could result in a number of positive patient outcomes.

A team, led by Cody P. Doolan, Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary, examined whether rapid near patient testing (NPT) reduced patient isolation time, hospital lengths of stay, antibiotic usage, and cost when there are suspected cases of CDI.

Standard of Care

Generally, when there are suspected cases of CDI in hospital settings, the reaction is patient isolation, laboratory testing, infection control, and presumptive treatment.

In the two-period pragmatic cluster randomized crossover trial, the investigators examined patients at 39 wards and divided them into 2 separate arms.

They sought primary outcomes of the effect of near patient testing on patient isolation time. To do this they used mixed effect generalized linear regression models.

The investigators also sought various secondary outcomes, including hospital length of stay and antibiotic therapy based on a negative binomial regression model.

Finally, they conducted natural experiment, intention-to-treat, and per protocol analyses.

The patient population included 656 patients who received NPT for CDI and 1667 patients in the standard of care cohort.

The Results

The results show a significant decrease in patient isolation time for the NPT group compared to the others (NE 9.4 hours; P <0.01, ITT, 2.3; P <0.05; PP, 6.7; P <0.1), as well as a significant reduction in hospital length of stay for short stays (NE, 47.4%; P <0.01; ITT, 18.4%; P <0.01; ITT, 34.2%; P <0.01).

For every additional hour of delay for a negative result there was an increase in metronidazole use (24 DDD per 1000 patients; P <0.05) and non-CDI treating antibiotics by 70.13 mg (P <0.01).

In the cost analysis, the investigators found NPT testing saved $25.48 per patient including test cost and patient isolation.

“The cluster randomized cross-over trial demonstrated that implementation of CDI NPT can contribute to significant reductions in isolation time, hospital length of stay, antibiotic usage, and health care cost,” the authors wrote.

CDI During COVID-19

Recently, investigators found prevalence of CDI in patients hospitalized with COVID-19 infections was relatively low during the pandemic.

In a study published in January, a team, led by Aalam Sohal, MBBS, Liver Institute Northwest, assessed the prevalence and impact of CDI in a population of hospitalized patients with COVID-19 infections in the US.

However, the study of 1.5 million patients show patients with CDI and COVID-19 were at an increased risk of negative outcomes, including mortality.

Overall, there was a higher incidence of mortality in the CDI group compared to patients without COVID-19 (23.25% vs. 13.33%; P <0.001).

Patients with COVID-19 and CDI had a higher incidence of sepsis (7.69% vs. 5%, P <0.001), shock (23.59% vs. 8.59%, P <0.001), ICU admission (25.54% vs. 12.28%, P <0.001), and AKI (47.71% vs. 28.52%, P <0.001).

The investigators found patients with CDII had a statistically significant higher risk of mortality (aOR, 1.47; P <0.001) compared to those without CDI after conducting a multivariable analysis.

There was also a statistically significant higher risk of sepsis (aOR, 1.47; P <0.001), shock (aOR, 2.7; P <0.001), AKI (aOR, 1.5; P <0.001), and ICU admission (aOR, 2.16; P <0.001).


Doolan CP, Sahragard B, Leal J, et al. Clostridioides difficile near patient testing versus centralized testing: A pragmatic cluster randomized cross-over trial. Clinical Infectious Diseases. 2023. doi:10.1093/cid/ciad046

New Pathogen Diagnostic Test is 1,000 Times More Sensitive than Conventional Tests

A team led by Srikanth Singamaneni has developed a new point-of-care diagnostic test that is 1,000 times more sensitive than conventional rapid tests and can quantify concentrations of proteins.

When Srikanth Singamaneni and Guy Genin, both professors of mechanical engineering & materials science in the McKelvey School of Engineering at Washington University in St. Louis, established a new collaboration with researchers from the School of Medicine in late 2019, they didn’t know the landscape of infectious disease research was about to shift dramatically. In a conference room overlooking Forest Park on a beautiful fall day, the team had one goal in mind: tackle the biggest infectious disease problem facing the world right then.

“Srikanth and I had a vision of a simple, quantitative diagnostic tool, so we connected with infectious disease physicians here at WashU and asked them, ‘What are the most important questions that could be answered if you could get really detailed information cheaply at the point of care?’” said Genin, the Harold and Kathleen Faught Professor of Mechanical Engineering. “Greg Storch told us that one of the most important challenges facing the field of infectious disease is finding a way to figure out quickly if a patient has a bacterial infection and should get antibiotics or has a viral infection for which antibiotics will not be effective.”

Storch, MD, the Ruth L. Siteman Professor of Pediatrics at the School of Medicine, was interested in diseases that affect most people regularly – colds, strep throat or the flu – but that weren’t getting as much research attention as rarer diseases. “Even with great advances that have been made in infectious disease diagnostics, there is still a niche for tests that are simple, rapid and sensitive,” Storch said. “It would be especially powerful if they could provide quantitative information. Tests with these characteristics could be employed in sophisticated laboratories or in the field.”

Research team members Ige George, MD, and Sumanth Gandra, MD, associate professors in the Division of Infectious Diseases at the School of Medicine, noted that the challenge is even greater in resource-limited settings.

Ultrasensitive and quantitative lateral flow assays have been created and tested through a collaboration between the McKelvey School of Engineering and the Washington University School of Medicine in St. Louis.  As in standard pregnancy or antigen tests, proteins within bodily fluids that are dripped onto the blue pad can be captured by labeled antibodies on the yellow pad as the fluid spreads down the strip. If the protein of interest is caught, the labels are snared by the green test strip before reaching the light blue control strip.  Unlike standard tests, the labels are plasmonic fluors. Whereas standard tests give just a yes/no answer about whether a protein of interest is present, these ultrabright particles can be imaged with a simple handheld device to quantify the amount of protein present.

In some of the clinics we work with in India, fast, simple and accurate diagnostic tests at the point of care continue to elude us,” George said. “For example, tuberculosis is one of the leading causes of death in poor countries, where up to one-third of patients are left undiagnosed. For other bacterial infections the costs associated with identifying bacteria and checking susceptibility to antibiotics exceeds the costs of prescribing antibiotics, leading to overuse of antibiotics.”

The problem is not confined to poor countries but also occurs here in St. Louis, added Gandra. “Take gonococcal infection, which has developed resistance to antibiotics over the years. At present, gonococcal infection cannot be diagnosed with simple and highly accurate point-of-care tests. This leads to treatment delays and infection spread,” Gandra said.

Drawing on his years of experience in developing nanomaterials for applications in biology and medicine, Singamaneni, the Lilyan & E. Lisle Hughes Professor, sought to overcome these limitations in point-of-care diagnostic tests. Singamaneni and his lab developed ultrabright fluorescent nanolabels called plasmonic-fluors, which could be quickly integrated into a common testing platform, the lateral flow assay (LFA). Plasmon-enhanced LFAs (p-LFAs) improve inexpensive, readily available rapid tests to levels of sensitivity required by physicians for confidence in test results without the need for lab-based confirmation.

“The high sensitivity and specificity of the novel biodiagnostic technology combined with its simplicity and low cost make it highly attractive for adoption in the clinical lab and point-of-care settings,” said team member Bijal Parikh, MD, PhD, assistant professor in the Department of Pathology & Immunology at Washington University School of Medicine. Parikh is also the medical director of the Molecular Diagnostics Laboratory.

According to findings published Feb. 2 in Nature Biomedical Engineering, the team’s p-LFAs are 1,000 times more sensitive than conventional LFAs, which show results via a visual color and fluorescence signal on the strip. When analyzed using a fluorescence scanner, p-LFAs are also substantially faster than gold-standard lab tests, returning results in only 20 minutes instead of several hours, with comparable or improved sensitivity. The p-LFAs can detect and quantify concentrations of proteins, enabling them to detect bacterial and viral infections as well as markers of inflammation that point to other diseases. 

“Plasmonic-fluors are composed of metal nanoparticles that serve as antennae to pull in the light and enhance the fluorescence emission of molecular fluorophores, thus making it an ultrabright nanoparticle,” Singamaneni explained. “Our p-LFAs can pick up even very small concentrations of antibodies and antigens, typical markers of infection, and give clinicians clear, quick results without the need for specialized equipment. For quantitative testing beyond the initial screening, the same LFA strip can be scanned with a fluorescence reader, enabling rapid and ultrasensitive colorimetric and fluorometric detection of disease markers with only one test.”

“It's like turning up the volume on standard color-changing test strips. Instead of getting a faint line indicating only a positive or negative result, the new p-LFAs give clearer results with fewer particles, enabling one to move from simply ‘yes or no?’ to exactly ‘how much?’ with the aid of an inexpensive, portable scanner,” said Jeremiah Morrissey, research professor in anesthesiology in the Division of Clinical and Translational Research at the School of Medicine. Morrissey is a co-author of the new study and long-term collaborator with the Singamaneni lab.

Co-author Pratik Sinha, MD, PhD, an intensive care and emergency medicine physician also in the Department of Anesthesiology in the Division of Clinical and Translational Research pointed to potential benefits of the new technology. “The LFA technology and inexpensive reader can be readily adapted to the intensive care unit or the emergency room where rapid determination of a patient’s medical condition can be used to tailor appropriate treatment and determine the efficacy of treatment,” Sinha said. “Given how relatively inexpensive it is compared to standard assays, the potential for this technology can be global.” 

This improved testing capability has obvious benefits for a population now all too familiar with the need for quick and reliable test results and the risk of false negatives.

“When we took on this problem in 2019, we thought our biggest challenge would be getting an adequate number of samples from sick people,” Genin recalled. “Where on Earth could we find a massive set of samples from patients whose symptoms were carefully documented and whose diagnosis was verified by slow and expensive PCR tests?” In a matter of months, COVID-19 would erase that obstacle while introducing a whole host of new challenges and opportunities.

“The pandemic was a big shift for us, like it was for everyone,” said first author Rohit Gupta, who worked on the p-LFA study as a graduate student in Singamaneni’s lab and is now a senior scientist at Pfizer. “We had to move away from our original focus on distinguishing viruses from bacteria, but it turned out to be an opportunity to do practical science with real stakes. We were working with epidemiologists to get samples for testing, with diagnosticians to compare our test to what was available, and with clinicians to gain insights into the real needs for patient care.”

Input from the entire collaboration helped Gupta and Singamaneni refine the design of the p-LFAs, which ultimately achieved 95% clinical sensitivity and 100% specificity for SARS-CoV-2 antibodies and antigens. Genin described the results as stunning. 

“We didn’t know it was going to work so well,” he said. “We knew it would be good, but we didn’t know this $1 test with a $300 readout device would be so much better – 10 times better – than state of the art that we all used during the COVID pandemic.”

Now that they’ve proven p-LFAs can outperform standard lab tests in sensitivity, speed, convenience and cost for one disease, the team is looking to develop new applications for the technology, including returning to their original goal of identifying bacterial versus viral infections and getting their diagnostic tool into the hands of physicians around the world.

The p-LFA technology has been licensed to Auragent Bioscience LLC by Washington University’s Office of Technology Management. Singamaneni and Morrissey are among the co-founders of Auragent, a WashU startup.

“We expect to have p-LFAs commercially available in the next one to two years,” Singamaneni said. “Right now, we’re working on improving our portable scanner technology, which adds a more sensitive, fluorescent reading capability to the test strips in addition to the color change that can be seen with the naked eye. We think we can get that cost down to a point where it’s accessible to rural clinics in the U.S. and abroad, which was one of our original goals.”

“We’re also excited about the potential to detect many more diseases than COVID, possibly using a skin patch that can take a painless sample,” Singamaneni added. “This technology has the potential to detect any number of diseases, ranging from STIs to respiratory infections and more, as well as cytokines indicative of inflammation seen in conditions such as rheumatoid arthritis and sepsis.” 

Gupta R, Gupta P, Wang S, Melnykov A, Jiang Q, Seth A, Wang Z, Morrissey JJ, George I, Gandra S, Sinha P, Storch GA, Parikh BA, Genin GM, Singamaneni S. “Plasmon-enhanced, quantitative lateral flow assay for femtomolar detection of protein biomarkers in point-of-care settings.” Nature Biomedical Engineering. Feb. 2, 2023. DOI: 10.1038/s41551-022-01001-1.

This research was supported by funding from the National Science Foundation (CBET-2027145, CBET-2029105, and CMMI 1548571); the National Cancer Institute-Innovative Molecular Analysis Technologies (R21CA236652 and R21CA236652-S1); and the Washington University Institute of Clinical and Translational Sciences (UL1TR002345) from the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health (NIH).

Researchers Propose Rapid Identification and Drug Resistance Screening of Respiratory Pathogens

In a study published in Frontiers in Microbiology, a cooperative research group from the Changchun Institute of Optics, Fine Mechanics and Physics (CIOMP) of the Chinese Academy of Sciences (CAS) and Jilin University proposed rapid identification and drug resistance screening of pathogenic bacteria based on single cell Raman spectroscopy (SCRS).

SCRS is a "whole biological fingerprint" technique that can be used to identify microbial samples. It can be used for in situ, non-invasive, and non-labeled detection of samples and has a great application value for qualitative analysis, quantitative analysis, and molecular structure determination.

The combination of D2O isotope labeling technology and SCRS can identify the Raman shift caused by the difference of metabolic activity between cells, which can be used as a semi-quantitative method to identify the metabolic activity of cells. The combination of the Raman and heavy water labeling techniques at the single-cell level can overcome the requirement of long-term culture in clinical pathogen experiments, making rapid drug screening possible.

In this study, a SCRS technique combined with a heavy water labeling was designed as a rapid and accurate method for detecting antibiotic-resistant bacteria in respiratory bacteria. And a Raman phenotype database of six common respiratory pathogens was established to provide a new method for rapidly identifying respiratory pathogens in clinical practice. Different antibiotic-heavy water labeling conditions were also explored to achieve drug resistance identification of respiratory pathogens within two hours.

Results showed the classification accuracy of the isolated samples was 93–100%, and the classification accuracy of the clinical samples was more than 80%. Combined with heavy water labeling technology, the drug resistance of respiratory tract pathogens was determined.

The study showed that SCRS–D2O labeling could rapidly identify drug resistance of respiratory tract pathogens within two hours, which provides a rapid and sensitive method for identifying respiratory tract infection bacteria and their drug susceptibility.


Ziyu Liu et al, Rapid identification and drug resistance screening of respiratory pathogens based on single-cell Raman spectroscopy, Frontiers in Microbiology (2023). DOI: 10.3389/fmicb.2023.1065173

Saturday, February 4, 2023

New Electrochemical Sensing Technology Enables Detection of SARS-CoV-2 Antigen-specific Antibodies

Not all SARS-CoV-2 infections are created equal. We have learned this through multiple virus waves are taking their toll on the world's population. Improving vaccines and new anti-viral therapies that target distinct viral molecules (antigens) and the changes they undergo over time have helped to soften this blow. However, to control the disease even better and everywhere, we have to be able to assess whether and with which viral variant individuals have been infected, what kind of protective immunity they possess, and how they respond to vaccinations and therapies.

An obvious way to accomplish this is through the detection of antibodies that the immune system produces against the virus' proteins and variant-specific antigens. Importantly, currently available COVID vaccines induce the production of antibodies against the Spike (S) protein, but rarely the N protein, while natural infection produces antibodies against both proteins. This allows the immune responses to be clearly distinguished from each other. Having a way to detect these different antibody types could inform health care and drug development decisions in a more systematic way. However, current antibody detection technologies are time-consuming, too costly, often require clinical laboratories, are and not able to accurately measure the levels of antibodies against multiple antigens, or they suffer from a combination of these inadequacies – which prevent them from being able to rapidly and effectively generate data about antibodies across global populations.

Now, an in-depth study from a research team at the Wyss Institute for Biologically Inspired Engineering at Harvard University demonstrates that the Institute's portable electrochemical sensing technology known as eRapid could be an ideal instrument to enable the inexpensive, multiplexed detection of different SARS-CoV-2-directed antibodies at the point-of-care. The team, led by Wyss Founding Director Donald Ingber, M.D., Ph.D. and Wyss Senior Scientist Pawan Jolly, Ph.D., showed that specifically engineered eRapid sensors can detect antibodies targeting the virus' so-called nucleocapsid (N) protein from ultra-small samples of blood plasma and dried blood spots with 100% sensitivity and specificity within less than 10 minutes. The findings are published in Biosensors and Bioelectronics.

Taking aim at COVID-induced immunity

"The study's findings further validate that our much-evolved version of the eRapid diagnostic technology is capable of a fast, accurate, and differentiated assessment of antibodies against viral antigens in individuals," said Ingber. "We can obtain these results at extremely low cost using extremely small samples that individuals could easily self-collect and test at home or send to central laboratories. Thus, eRapid opens the opportunity of being used as a tool for pandemic surveillance and therapeutic monitoring, not only in the present but also for future pandemic and epidemic outbreaks." Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children's Hospital, and the Hansjörg Wyss Professor of Bioinspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS).

The new findings build on a previous study that showed eRapid technology is capable of simultaneously detecting SARS-CoV-2-specific RNA and antibodies on the same electrochemical sensor chips. In their new study, the team honed further in on the N protein and virus-induced immunity. Using specifically engineered eRapid sensors and a collection of 93 clinical samples of only 1.5 microliters in volume, they were able to distinguish 54 SARS-CoV-2 positive patients from 39 negative individuals within 10 minutes, with 100% sensitivity (all positive samples identified) and 100% specificity (all negative samples identified).

New diagnostic possibilities

"The combined features of eRapid make it an extremely useful platform for the fast and multiplexed detection of antibodies emerging in patients against a growing and fluctuating number of viral and other antigens, and for following an individual's antibody levels over time as we showed in our new study," said Jolly. "We took the Wyss' eRapid platform through an extensive de-risking program by engineering new nanochemistry, manufacturing, and sensing abilities. At this point, we'd like to see our technology benefit as many patients in as many disease areas as possible, including, of course, infectious diseases such as COVID-19."

In 2022, the eRapid technology was licensed to the Wyss' startup StataDX for the fields of neurological, cardiovascular, and renal diseases. First author Sanjay Sharma Timilsina, Ph.D., and second author Nolan Durr, two former members of the Wyss' eRapid team who had been instrumental in advancing the novel electrochemical sensing approach as a diagnostic platform, joined StataDX. The Wyss Institute is currently exploring additional commercial opportunities to commercialize eRapid for multiple other application areas including infectious disease diagnostics.

"With strides that we are making in parallel on developing portable devices for housing eRapid diagnostic assays, we believe that eRapid could serve as one of the first multiplexed diagnostic platforms for a wide variety of diagnostic applications as it is based on electrochemical detection and so functions much like the glucometer that is already used world-wide for patients with diabetes," said Ingber.

The study was funded by the Wyss Institute for Biologically Inspired Engineering at Harvard University and GBS Inc.

Source: Wyss Institute for Biologically Inspired Engineering at Harvard


Timilsina, S.S., et al. (2022) Rapid quantitation of SARS-CoV-2 antibodies in clinical samples with an electrochemical sensor. Biosensors and Bioelectronics.