Tuesday, June 21, 2022

Rapid Ebola Diagnosis May Be Possible With New Technology

A new tool can quickly and reliably identify the presence of Ebola virus in blood samples, according to a study by researchers at Washington University School of Medicine in St. Louis and colleagues at other institutions.

The technology, which uses so-called optical microring resonators, potentially could be developed into a rapid diagnostic test for the deadly Ebola virus disease, which kills up to 89% of infected people. Since it was discovered in 1976, Ebola virus has caused dozens of outbreaks, mostly in central and west Africa. Most notable was an outbreak that began in 2014 and killed more than 11,000 people in Guinea, Sierra Leone and Liberia; in the U.S., the virus caused 11 cases and two deaths. A rapid, early diagnostic could help public health workers track the virus’ spread and implement strategies to limit outbreaks.

The study — which also involved researchers from the University of Michigan, Ann Arbor, and Integrated Biotherapeutics, a biotech company — is published June 8 in Cell Reports Methods.

“Any time you can diagnose an infection earlier, you can allocate health-care resources more efficiently and promote better outcomes for the individual and the community,” said co-first author Abraham Qavi, MD, PhD, a postdoctoral researcher at Washington University. “Using a biomarker of Ebola infection, we’ve shown that we can detect Ebola infection in the crucial early days after infection. A few days makes a big difference in terms of getting people the medical care they need and breaking the cycle of transmission.”

Ebola virus is transmitted by contact with bodily fluids. It causes fever, body aches, diarrhea and bleeding — nonspecific symptoms that easily can be mistaken for other viral infections or for malaria. In recent years, vaccines and effective therapies for Ebola have been developed, but they are not widely available. Instead, health officials control the deadly virus by containing outbreaks. The strategy relies on quickly identifying infected people and preventing transmission by encouraging caregivers to wear protective gear.

Qavi had previously worked with Ryan C. Bailey, PhD, the Robert A. Gregg Professor of Chemistry at the University of Michigan and a co-senior author on this paper, to co-develop optical microring resonators, a kind of whispering gallery mode device used for molecular detection. The name comes from the Whispering Gallery at St. Paul’s Cathedral in London. A whisper uttered on a walkway in the dome above the nave can be heard clearly more than 100 feet away because the sound waves increase in amplitude as they bounce around the circular wall. The 18th century builders accidentally constructed a giant demonstration of the principle of acoustic resonance, in which sound waves increase in amplitude if they interact in precisely the right way. The same phenomenon occurs with light waves on a much smaller scale.

When Qavi joined the lab of co-senior author Gaya K. Amarasinghe, PhD — an Ebola expert and the Alumni Endowed Professor of Pathology & Immunology and a professor of biochemistry & molecular biophysics and of molecular microbiology at Washington University — they decided to apply the technology to create a better diagnostic test for Ebola. Qavi teamed up with Bailey, co-first author Krista Meserve, a graduate student in Bailey’s lab, and co-author Lan Yang, PhD, the Edwin H. and Florence G. Skinner Professor of Electrical & Systems Engineering at Washington University’s McKelvey School of Engineering, to develop a tool that could detect tiny amounts of Ebola-related molecules in blood samples using microring resonators.

“We trap light in the resonators and use resonance to enhance and boost our signal,” Qavi said. “By monitoring where this resonance wavelength occurs, we can tell how much of the molecule we have.”

The key was finding the right molecule. Current diagnostic tests detect the virus’s genetic material or a glycoprotein — a protein covered in sugar — produced by the virus. But they aren’t reliable until the virus has multiplied to high levels in the body, a process that can take days. Co-senior author Frederick Holtsberg, PhD, vice president of manufacturing and bioanalytics at Integrated Biotherapeutics, developed a highly sensitive antibody capable of detecting the viral soluble glycoprotein at low levels.

The researchers incorporated the antibody into their device and tested it using blood from infected animals. They found their technique could detect the glycoprotein as early as or earlier than the most sensitive test for viral genetic material. Importantly, the technology also allowed them to quantify the amount of viral glycoprotein in the blood. The higher the level, the worse the infected animals fared. Moreover, the test only took 40 minutes start to finish.

“Looking at these data, we can say, ‘If you’re above these levels, your chance of survival is low; if you’re below it, your chance of survival is high’,” Qavi said. “We still have to validate this in infected individuals, but if it holds up, doctors could use this information to tailor treatment plans for individual patients and allocate scarce medications to the patients most likely to benefit.

“We’ve shown the fundamental science works,” he added. “Now it’s just an issue of miniaturizing the devices and taking them into the field.”

Monday, June 20, 2022

Raman Spectroscopy as an Alternative to the Conventional Sterility Test

A study combining Raman spectroscopy with PLS-DA multivariate analysis achieved fast and non-invasive detection of contaminated drug products within vials.

Researchers have demonstrated the potential of a fast and non-invasive approach to detect pharmaceutical products contaminated with low levels of bacteria within their vials. The technique uses dispersive Raman spectroscopy (RS) in association with partial least squared discriminant analysis (PLS-DA).

Sterility testing is a crucial step in quality control of pharmaceutical drug products before their commercial release. However, the current procedures for bioburden testing, which are primarily growth based, are highly time-consuming, costly and have limited sensitivity and specificity.

RS is under investigation as a lower cost, more rapid alternative; however, researchers have struggled to balance robustness, sensitivity, cost and a low limit of detection (LOD).

According to a new study, currently available in pre-print at bioRxiv, key challenges that new techniques must overcome include:

(a) discrimination between Raman spectra from organic molecules in the formula and bacterial ones
(b) detection at low contamination, overcoming the weak signal from bacteria
(c) contribution to the Raman signal from other sources such as product packaging, fluorescent compounds
(d) correct data processing and statistical analysis model.

The study presents an approach to up-concentrate and detect ≤10 colony forming units (CFU)/ml of relevant bacteria with RS and multivariate analysis without breaching the primary drug product package.

To increase the Raman signal and the likelihood contaminants would be detected, the vials were centrifuged in an inclined (upside-down) position to localize the bacteria close to the neck of the product vial.

The RS-PLS-DA approach enabled the fast and non-invasive discrimination of products vials containing low numbers of bacteria from sterile ones without breaching the packaging. The technique enabled the identification of three different bacteria Bacillus subtilis, Salmonella enterica and Staphylococcus haemolyticus, as well as B. subtilis spores with an accuracy of 99 percent. The method was able to distinguish samples with vegetative cells and spores in limits <10 CFU/ml, even in the presence of other organic molecules from the product formula in the container.

Independent validation was able to confirm the high sensitivity and specificity.

According to Grosso et al., the project supports the use of the RS-PLS-DA approach as alternative to the pharmacopeial destructive sterility testing method. “We provide a feasible approach using RS in association with PLS-DA to detect extremely low numbers of cells or spores with high accuracy and reproducibility without compromising the robustness of the method,” wrote the authors. “These results support Raman spectroscopy as a promising biotechnological tool suitable for bioburden test in quality control of pharmaceutical industry.”

They added that the RS-PLS-DA method developed could enable real-time monitoring of contamination in pharmaceutical processing.

Tuesday, June 7, 2022

New Blood Test Can Help Doctors Diagnose Tuberculosis and Monitor Treatment

Researchers at Tulane University School of Medicine have developed a new highly sensitive blood test for tuberculosis (TB) that screens for DNA fragments of the Mycobacterium tuberculosis bacteria that causes the deadly disease.

The test could give doctors a new tool to both quickly identify TB and then gauge whether drug treatments are effective by monitoring levels of DNA from the pathogen circulating through the bloodstream, according to a new study published in the journal The Lancet Microbe.

Tuberculosis is now the second most deadly infectious disease in the world, behind only COVID-19. In 2020, an estimated 10 million people contracted TB and 1.5 million people died from it, according to the World Health Organization. 

Most TB tests rely on screening sputum, a thick type of mucus from the lungs. But collecting sputum from patients suspected of having TB can be difficult, especially for children. TB can also be harder to diagnose in immunocompromised HIV patients and others where the infection migrates outside of the lungs into other areas of the body. In these extrapulmonary cases, patients can have little bacteria in the sputum, which leads to false negatives using current testing methods, said lead study author Tony Hu, PhD, Weatherhead Presidential Chair in Biotechnology Innovation at Tulane University.

“This assay may be a game-changer for TB diagnoses that not only provides accurate diagnosis results but also has the potential to predict disease progression and monitor treatment,” Hu said. “This will help doctors rapidly intervene in treatment and reduce the risk of death, especially for children living with HIV.”

The study evaluated a CRISPR-based assay that screened for cell-free DNA from live Mycobacterium tuberculosis bacilli. The screening target is released into the bloodstream and cleared quite rapidly, providing a real-time snapshot of active infection.

Researchers tested preserved blood samples from 73 adults and children with presumptive TB and their asymptomatic household contacts in Eswatini, Africa. 

The test identified adult TB with 96.4% sensitivity and 94.1% specificity and pediatric TB with 83.3% sensitivity and 95.5% specificity. (Sensitivity refers to how well a test can diagnose a positive case, while specificity is a measure of a test’s accurately determining a negative case.)

Researchers also tested 153 blood samples from a cohort of hospitalized children in Kenya. These were HIV-positive patients who were at high risk for TB and presented with at least one symptom of the disease. The new test picked up all 13 confirmed TB cases and almost 85% of unconfirmed cases, which were cases that were diagnosed due to clinical symptoms and not existing gold standard testing methods.

The CRISPR-based test uses a small blood sample and can deliver results within two hours.

“We are particularly excited that the level of Mycobacterium tuberculosis cell-free DNA in HIV-infected children began to decline within a month of treatment, and most of the children's blood was cleared of the bacteria DNA fragments after treatment, which means that CRISPR-TB has the potential to monitor treatment and will give physicians the ability to better treat worldwide TB infections,” Hu said.

The researchers have since adapted the assay to a rapid test platform that can deliver results in 30 minutes without any special equipment. Results would be viewable on a paper strip like a rapid COVID-19 test.  

“A highly accurate, rapid blood test that could be used anywhere would benefit millions of people living in resource-limited areas with a high TB burden,” Hu said.

The full results of the paper are online here

Source: Tulane University School of Medicine 

Portable Sensor Technology Aims to Quickly Detect Foodborne Contaminants Outside the Lab

An international team led by a University of Massachusetts Amherst food and environmental virologist has received a $750,000 USDA National Institute of Food and Agriculture (NIFA) partnership grant to develop and test portable, rapid biosensors capable of detecting noroviruses and mycotoxins in foods and agricultural products. It is among the first partnership grants awarded with an international partner by the USDA.

Noroviruses are the leading cause of foodborne illness globally, and are highly contagious, causing pandemics every few years, says lead investigator Matthew Moore, assistant professor of food science. Moore will work with UMass Amherst food science colleague John Gibbons, a fungi expert, and food science Ph.D. candidate Sloane Stoufer in the Moore Lab. The UMass team will collaborate with senior lecturer and principal investigator Marloes Peeters and postdoctoral research associate Jake McClements at Newcastle University’s School of Engineering in England.

“People can get really sick from foods that contain viruses and toxins,” Moore says. “We need a way to quickly and easily find out if a food contains these contaminants in a cheap but effective way – without the need to go back to a separate lab to do the testing.”

Mycotoxins are toxic substances produced by fungi that can grow in warm and humid conditions on crops and food, in particular in many grains, produce, nuts, seeds and spices. They represent a growing threat to public health in the face of climate change trends and increased consumption of plant-based foods, Moore says.

“One of the interesting things about mycotoxins as a foodborne contaminant is that they’re often not very acute, so you’re less likely to notice it,” Moore says. “Oftentimes, the damage they do is more chronic, and they will mess with the kidneys and liver especially and can promote cancer.”

That makes early detection all the more important. “With this technology we’re trying to create a cheap, highly durable, and potentially reusable sensor that can detect these contaminants,” Moore says.

The UMass Amherst food scientists got together with engineers at Newcastle University to seek a rare international partnership grant from the USDA’s NIFA. The British engineers are world leaders in electrochemical sensing techniques based on generating molecularly imprinted polymer nanoparticles (nanoMIPs).

“The grant enables an unprecedented international exchange,” Moore says. The UMass team will learn more about the application of nanoMIPs when they visit the Peeters Lab at Newcastle, and the UK team will be hosted by Moore’s Applied and Environmental Virology Lab to gain knowledge about virological, microbiological and food science techniques.

“This nanoMIP-based sensing technology has numerous advantages,” Moore says. “It is very stable in intense conditions, and very portable. It is also quite inexpensive, a very important consideration in testing for foods.”

NanoMIP-based electrochemical sensing is an exciting new application for agricultural targets. “The technology has already shown promise for other targets, including SARS-CoV-2, and we hope to further explore its potential for human noroviruses and mycotoxins,” Moore says.

Source: University of Massachusetts Amherst