Electric stimulation may be able to help blood vessels carry white blood cells and oxygen to wounds, speeding healing, a new study suggests.
The study, published in the Royal Society of Chemistry journal Lab on a Chip, found that steady electrical stimulation generates increased permeability across blood vessels, providing new insight into the ways new blood vessels might grow.
The electrical stimulation provided a constant voltage with an accompanying electric current in the presence of fluid flow. The findings indicate that stimulation increases permeability of the blood vessel — an important characteristic that can help wound-healing substances in the blood reach injuries more efficiently.
“There was this speculation that blood vessels could grow better if you stimulated them electrically,” said Shaurya Prakash, senior author of the study and associate professor of mechanical and aerospace engineering at The Ohio State University. “And we found that the response of the cells in our blood vessel models shows significant promise towards changing the permeability of the vessels that can have positive outcomes for our ongoing work in wound healing.”
Blood vessels are crucial for wound healing: They thread throughout your body, carrying nutrients, cells and chemicals that can help control inflammation caused by an injury. Oxygen and white blood cells — which protect the body from foreign invaders — are two key components delivered by blood vessels.
But when there is an injury — for example, a cut on your finger — the architecture of the blood vessels at the wound site are disrupted. That also interrupts the vessels’ ability to help the wound heal. Blood vessels regrow on their own, almost like the branches of trees, without external sources of electricity, as part of the healing process.

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“And as the blood vessels begin to grow, they replenish the skin and cells and establish a healing barrier again,” Prakash said. “But our question was: How do you make this process better and faster, and is there any benefit to doing that?”
What they found, in laboratory tests performed using human cells, is that stimulating blood vessels with electricity showed a marked increase in blood vessel permeability, which is a physical marker suggestive of possible new vessel growth.
“These initial findings are exciting, and the next phase of the work will require us to study if and how we can actually grow new vessels,” Prakash said.
Jon Song, co-author of the paper and associate professor of mechanical and aerospace engineering at Ohio State, said the results imply that one of the primary ways blood vessels work to heal injuries is by allowing molecules and cells to move across the vessels’ walls.
“And now we have better understanding for how electric stimulation can change the permeability across the vessel walls,” Song said. “Let’s say you have a cutaneous wound, like a paper cut, and your blood vessels are severed and that’s why you have blood leaking out. What you need is a bunch of bloodborne cells to come to that place and exit out the blood vessel to initiate the wound repair.”
The study suggested that changes in blood vessel permeability could get those bloodborne cells to a wound site more quickly, though it did not explain the reasons why that happened. The study seemed to indicate that electricity affected the proteins that hold blood vessel cells together, but those results were not conclusive.
The study is an extension of work by a broader team, led by Prakash, that previously showed electric bandages could help stimulate healing in wounded dogs. That work indicated that electrical stimulation might also help manage infections at wound sites — a phenomenon the researchers also hope to research further.

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Materials provided by Ohio State University. Original written by Laura Arenschield. Note: Content may be edited for style and length.

Millions of people die prematurely every year from diseases and cancer caused by air pollution. The first line of defence against this carnage is ambient air quality standards. Yet, according to researchers from McGill University, over half of the world’s population lives without the protection of adequate air quality standards.
Air pollution varies greatly in different parts of the world. But what about the primary weapons against it? To find answers, researchers from McGill University set out to investigate global air quality standards in a study published in the Bulletin of the World Health Organization.
The researchers focused on air pollution called PM2.5 — responsible for an estimated 4.2 million premature deaths every year globally. This includes over a million deaths in China, over half a million in India, almost 200,000 in Europe, and over 50,000 in the United States.
“In Canada, about 5,900 people die every year from air pollution, according to estimates from Health Canada. Air pollution kills almost as many Canadians every three years as COVID-19 killed to date,” says co-author Parisa Ariya, a Professor in the Department of Chemistry at McGill University.
Small but deadly
Among the different types of air pollution, PM2.5 kills the most people worldwide. It consists of particles smaller than approximately 2.5 microns — so small that billions of them can fit inside a red blood cell.
“We adopted unprecedented measures to protect people from COVID-19, yet we don’t do enough to avoid the millions of preventable deaths caused by air pollution every year,” says Yevgen Nazarenko, a Research Associate at McGill University who conducted the study with Devendra Pal under the supervision of Professor Ariya.
The researchers found that where there is protection, standards are often much worse than what the World Health Organization considers safe. Many regions with the most air pollution don’t even measure PM2.5 air pollution, like the Middle East. They also found that the weakest air quality standards are often violated, particularly in countries like China and India. In contrast, the strictest standards are often met, in places like Canada and Australia.
Surprisingly, the researchers discovered that high population density is not necessarily a barrier to fighting air pollution successfully. Several jurisdictions with densely populated areas were successful in setting and enforcing strict standards. These included Japan, Taiwan, Singapore, El Salvador, Trinidad and Tobago, and the Dominican Republic.
“Our findings show that more than half of the world urgently needs protection in the form of adequate PM2.5 ambient air quality standards. Putting these standards in place everywhere will save countless lives. And where standards are already in place, they should be harmonized globally,” says Nazarenko.
“Even in developed countries, we must work harder to clean up our air to save hundreds of thousands of lives every year,” he says.

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Materials provided by McGill University. Note: Content may be edited for style and length.

Ten days after receiving a second dose of a messenger RNA, or mRNA, vaccine for COVID-19, patients without COVID-19 symptoms are far less likely to test positive and unknowingly spread COVID-19, compared to patients who have not been vaccinated for COVID-19. The Pfizer-BioNTech and Moderna messenger RNA vaccines for COVID-19 are authorized for emergency use in the U.S.
With two doses of a messenger RNA COVID-19 vaccine, people with no symptoms showed an 80% lower adjusted risk of testing positive for COVID-19 after their last dose. Those are the findings of a Mayo Clinic study of vaccinated patients. These finding appear in the journal Clinical Infectious Diseases.
The authors say these findings underscore the efficacy of messenger RNA vaccines for COVID-19 to significantly limit the spread of COVID-19 by people with no symptoms who may unknowingly spread the infection to others.
The researchers retrospectively looked at a cohort of 39,000 patients who underwent pre-procedural molecular screening tests for COVID-19. More than 48,000 screening tests were performed, including 3,000 screening tests on patients who had received at least one dose of a messenger RNA COVID-19 vaccine. These screening tests were part of routine COVID-19 testing prior to treatments not related to COVID-19, such as surgeries and other procedures. Patients in the vaccinated group had received at least one dose of a messenger RNA COVID-19 vaccine.
“We found that those patients without symptoms receiving at least one dose of the first authorized mRNA COVID-19 vaccine, Pfizer-BioNTech, 10 days or more prior to screening were 72% less likely to test positive,” says Aaron Tande, M.D., a Mayo Clinic infectious diseases specialist and co-first author of the paper. “Those receiving two doses were 73% less likely, compared to the unvaccinated group.”
After adjusting for a range of factors, researchers found an 80% risk reduction of testing positive for COVID-19 among those with two doses of a messenger RNA COVID-19 vaccine.
The study was based on patients receiving screening tests between Dec. 17, 2020, and Feb. 8 at Mayo Clinic in Minnesota and Arizona and at Mayo Clinic Health System in Minnesota and Wisconsin.
Additional authors are Benjamin Pollock, Ph.D., co-first author; Nilay Shah, Ph.D.; Gianrico Farrugia, M.D.; Abinash Virk, M.D.; Melanie Swift, M.D.; Laura Breeher, M.D.; Matthew Binnicker, Ph.D.; and Elie Berbari, M.D. ? all of Mayo Clinic. Funding for the study was provided by Mayo Clinic.

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Materials provided by Mayo Clinic. Original written by Robert Nellis. Note: Content may be edited for style and length.

Scientists have discovered a route of introduction for High Pathogenicity Avian Influenza Virus (HPAIV) H5N8 into Japan and, in parallel, have investigated the potential of two human anti-influenza drugs for the control of HPAI in birds.
Since October 30, 2020, there have been over 30 recorded outbreaks of High Pathogenicity Avian Influenza (HPAI) in domestic poultry and wild fowl in Japan. This outbreak was caused by the influenza A virus H5N8, a known High Pathogenicity Avian Influenza Virus (HPAIV). In such a scenario, identification of the source of the virus and its transmission route is important to control its spread.
A team of scientists led by Professor Yoshihiro Sakoda of Hokkaido University have recently found the probable route of introduction of the current HPAIV into Japan — by migratory birds from Europe. Separately, they showed that anti-influenza drugs used for humans can potentially be used to treat HPAI in poultry and wild fowl, providing an alternative to culling infected birds. Their findings were published within a week of each other in the journal Viruses.
HPAI is a devastating disease in poultry, leading to large losses both economically and materially. Once present in domesticated poultry, the primary means of controlling HPAI is by culling all infected populations. There is no approved drug for the treatment of HPAI. In addition, it can infect captive wild birds, such as those in zoos and sanctuaries, which has major implications for the protection and conservation of endangered species.
In addition, HPAI is closely related to influenza in humans; certain strains of HPAIV have jumped to humans in the past, most recently in mid February 2021, in Russia. For prevention and control, it is vital to track the spread of this disease.
The scientists collected migratory duck feces samples from the lakeside of Lake Komuke, eastern Hokkaido in October 2020. After a number of tests, they confirmed the presence of H5N8 virus in one of the samples. Further, their genetic analysis showed that the H5N8 virus was closely related to the variants that caused outbreaks in Europe from late 2019 to early 2020 and the variants found in Korea and southern Japan from October to November 2020, rather than from the H5N8 viruses in East Asia from 2018-2019. This suggested that the H5N8 virus transmitted with migratory birds from Europe to Eastern Asia within 10 months. In addition, the team found that it is a different H5N8 variant that is causing current outbreaks in Europe, raising the alarm that the northern biosphere is becoming a reservoir of HPAIV.
The scientists also investigated two antivirals, baloxavir marboxil (BXM) and peramivir (PR), used for the treatment of influenza in humans for their potential to treat HPAI in poultry. In their experiments, both drugs improved the survival rate of infected chickens and reduced viral amounts in their organs and feces, with BXM showing higher efficacy. Further work on BXM suggested that an early single-administration of BXM at doses of 2.5 mg/kg or higher would be most effective for the treatment of HPAI in real-life settings.
“Based on our findings, the government authorities warned poultries in Japan in November last year, which helped local businesses take measures to prevent potential outbreaks. As in the past years, we will continue to monitor HPAIV in migratory birds visiting Hokkaido as well as researching possible treatments of the disease,” said Sakoda.
The next steps would be to confirm if the strain of H5N8 detected by the scientists is responsible for the ongoing HPAI outbreak in Japan, and to verify if BXM is capable of treating HPAI in rare wild birds and poultry farms.

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Materials provided by Hokkaido University. Note: Content may be edited for style and length.

The genetics of human eye colour is much more complex than previously thought, according to a new study published today.
An international team of researchers led by King’s College London and Erasmus University Medical Center Rotterdam have identified 50 new genes for eye colour in the largest genetic study of its kind to date. The study, published today in Science Advances, involved the genetic analysis of almost 195,000 people across Europe and Asia.
These findings will help to improve the understanding of eye diseases such as pigmentary glaucoma and ocular albinism, where eye pigment levels play a role.
In addition, the team found that eye colour in Asians with different shades of brown is genetically similar to eye colour in Europeans ranging from dark brown to light blue.
This study builds on previous research in which scientists had identified a dozen genes linked to eye colour, believing there to be many more. Previously, scientists thought that variation in eye colour was controlled by one or two genes only, with brown eyes dominant over blue eyes.
Co-senior author Dr Pirro Hysi, King’s College London, said: “The findings are exciting because they bring us to a step closer to understanding the genes that cause one of the most striking features of the human faces, which has mystified generations throughout our history. This will improve our understanding of many diseases that we know are associated with specific pigmentation levels.”
Co-senior author Dr Manfred Kayser, Erasmus University Medical Center Rotterdam, said:
“This study delivers the genetic knowledge needed to improve eye colour prediction from DNA as already applied in anthropological and forensic studies, but with limited accuracy for the non-brown and non-blue eye colours.”

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Materials provided by King’s College London. Note: Content may be edited for style and length.

Extremely preterm infants can suffer from a life-threatening inflammation of the gut. A new clinical study has shown that supplements of a lactic acid bacterium may have positive effects by increasing the diversity of intestinal bacteria in these infants. The study has been led by researchers at Linköping University, Sweden, and published in the scientific journal Cell Reports Medicine.
A litre of milk weighs a kilogram. Most infants who are born extremely prematurely weigh less than that. An infant who should have developed and grown for three more months in the protective environment of the mother’s womb is, of course, extremely vulnerable. As a consequence of advances in neonatal care, many premature infants survive, although one out of four of the extremely premature infants die.
“Preterm infants can be affected by a very severe inflammation of the intestines, which almost only occurs in such infants. The condition, necrotising enterocolitis (or NEC), leads to parts of the intestine dying. One of three infants who contract the infection die, and those who survive often suffer from long-term complications such as short gut syndrome and neurodevelopmental disabilities,” says Thomas Abrahamsson, paediatrician at the neonatal intensive care unit at Crown Princess Victoria Children´s Hospital and associate professor at the Department of Biomedical and Clinical Sciences (BKV) at Linköping University, who has led the study.
The bacteria in the intestine of preterm infants differ from those in full-term infants. This has led many people to investigate whether giving probiotic supplements that contain certain bacteria has a positive effect. One finding is that the lactic acid bacterium Lactobacillus reuteri can reduce the risk of NEC in preterm infants. It is, however, not clear whether this is true also for extremely preterm infants, nor is the mechanism behind the positive effect known.
The study now published is part of a clinical study carried out in Linköping and Stockholm. The researchers looked at 132 infants who had been born extremely prematurely, between week 23 and 28 of pregnancy, i.e. 17 to 12 weeks before the due date. All weighed less than a kilogram at birth. Each infant was randomly assigned to one of two groups: to receive oil drops that contained the probiotic or placebo. The treatment was given daily during the neonatal period. The scientists investigated how the intestinal bacterial flora was influenced by the supplement of L. reuteri, and analysed bacteria in the stools at several time points.
“We see that the composition of bacteria in the intestine differs during the first month of the probiotic treatment. During the first week of life, the bacterial groups Staphylococcus and Klebsiella, which may cause disease, were more common in the group that received placebo,” says Magalí Martí Generó, principal research engineer in BKV, and principal author of the article.
Klebsiella can cause inflammation and has been associated with NEC and sepsis. The present study does not allow any conclusions about whether the probiotic treatment influences the risk of these diseases in these extremely premature infants. Larger studies will be required to determine this.
“The supplemented probiotic L. reuteri survives in the intestine, even though these extremely premature infants are treated with large doses of antibiotics that kill bacteria. The positive effect of the treatment in increasing the diversity of intestinal bacteria may be one mechanism behind the positive effects of this probiotic shown in previous studies,” says Thomas Abrahamsson.
Supplementation with probiotics is used in increasing numbers of neonatal clinics. The scientific evidence that supplements of probiotics to preterm infants have a positive effect and can be used safely is considered to be sufficiently strong.

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Materials provided by Linköping University. Original written by Karin Söderlund Leifler. Note: Content may be edited for style and length.

Patients suffering from dry eye disease symptoms have a lower quality of life compared to those without symptoms, a new study reports. The findings showed that patients with the condition reported negative effects on visual function, their ability to carry out daily activities and their work productivity.
Dry eye disease is a common condition and a frequent reason for patients to seek medical care. It can affect people of any age but is most prevalent in women and in older people. Symptoms include irritation and redness in the eyes, blurred vision, and a sensation of grittiness or a foreign body in the eye. It has been reported that up to a third of adults over 65 years old have the condition, although the actual number is likely to be higher as there is no established diagnostic test and people with mild symptoms are less likely to report them to their doctor.
Treatment often involves prescriptions of artificial tears, ocular lubricants and astringents, which come at a cost to the NHS; in 2014, 6.4 million items were prescribed at a cost of over £27 million.
This new study, led by the University of Southampton, set out to explore how dry eye disease affects the lives of adults in the UK through an online survey of one thousand patients with the condition and further one thousand without. Participants undertook a questionnaire from the National Eye Institute about their visual function and a EuroQol questionnaire on health-related quality of life. Those who declared that they experienced dry eye disease also answered further questions to assess the severity of their symptoms.
The results, published in the journal BMJ Open, showed that a higher proportion of participants with dry eye disease had problems with mobility and experienced more difficulties in their day-to-day activities than patients without the condition. The surveys also revealed they were more likely to suffer from anxiety and depression.
Those with the most severe symptoms we more likely to experience a negative impact on their social and emotional functioning as well work productivity, including missing more time from work as a result of their symptoms.
Dr Parwez Hossain, Associate Professor in Ophthalmology at the University of Southampton, led the study. He said: “This study provided some very useful information on the burden that dry eye disease places on patients. As well as confirming the impact on work and social lives we also discovered showed that the extent of the effects are consistent with the severity of symptoms. We also found that participants with dry eye disease symptoms were a lot more likely to suffer from other comorbidities, twice as many suffered from arthritis, hearing loss or irritable bowel disease compared to the cohort without symptoms.
“Whilst we cannot draw causal associations through this study, the presence of dry eye disease does appear to impact on an individual’s health and vision related quality of life.”
Although both groups reported similar levels of digital screen use and reading, the cohort with symptoms reported more exposure to environmental factors such as air conditioning, forced heating or air pollution. The research team believe that these factors could either contribute to dry eye disease, or be noticed more by sufferers.

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Materials provided by University of Southampton. Note: Content may be edited for style and length.

Researchers have developed a noninvasive technique that can capture images of rod and cone photoreceptors with unprecedented detail. The advance could lead to new treatments and earlier detection for retinal diseases such as macular degeneration, a leading cause of vision loss.
“We are hopeful that this technique will better reveal subtle changes in the size, shape and distribution of rod and cone photoreceptors in diseases that affect the retina,” said research team leader Johnny Tam from the National Eye Institute. “Figuring out what happens to these cells before they are lost is an important step toward developing earlier interventions to treat and prevent blindness.”
In Optica, The Optical Society’s (OSA) journal for high impact research, the researchers show that their new imaging method overcomes resolution limitations imposed by the diffraction barrier of light. The researchers accomplish this feat while using light that is safe for imaging the living human eye.
“The diffraction limit of light can now be routinely surpassed in microscopy, which has revolutionized biological research,” said Tam. “Our work represents a first step toward routine sub-diffraction imaging of cells in the human body.”
Using less light to see more
Achieving high-resolution images of photoreceptors in the back of the eye is challenging because the eye’s optical elements (such as lens and cornea) distort light in a way that can substantially reduce image resolution. The diffraction barrier of light also limits the ability of optical instruments to distinguish between two objects that are too close together. Although there are various methods for imaging beyond the diffraction limit, most of these approaches use too much light to safely image living human eyes.

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To overcome these challenges, the researchers improved upon a retinal imaging technique known as adaptive optics scanning light ophthalmoscopy, which uses deformable mirrors and computational methods to correct for optical imperfections of the eye in real time.
“One might think that more light is needed to get a better image, but we demonstrate that we can improve resolution by strategically blocking light in various locations within our instrument,” said Tam. “This approach reduces the overall power of light delivered to the eye, making it ideal for live imaging applications.”
For the new approach, the researchers generated a ring-shaped, or hollow, beam of light. Using this type of beam improved the resolution across the photoreceptors but at the expense of depth resolution. To regain the lost depth resolution, the researchers used a small pinhole called a sub-Airy disk to block light coming back from the eye. They showed that this imaging approach could be used to enhance a microscopy technique called non-confocal split-detection, which is used to acquire complementary views of the photoreceptors.
Testing in the clinic
After demonstrating that imaging resolution was improved in theoretical simulations, the researchers confirmed their simulations using various test targets. They then used the new method to image rod and cone photoreceptors in five healthy volunteers at the National Institutes of Health’s Clinical Center.
The new approach yielded about a 33 percent increase in transverse resolution and 13 percent improvement in axial resolution compared to traditional adaptive optics scanning light ophthalmoscopy. Using their optimized approach, the researchers were able to see a circularly shaped subcellular structure in the center of cone photoreceptors that could not be clearly visualized previously.
“The ability to noninvasively image photoreceptors with subcellular resolution can be used to track how individual cells change over time,” said Tam. “For example, watching a cell begin to degenerate, and then possibly recover, will be an important advance for testing new treatments to prevent blindness.”
The researchers plan to image the eyes of more patients with the new technique and use the images to begin to answer fundamental questions linked to rod and cone health. For example, they are interested in visualizing rod and cone health in people who have rare genetic diseases. They say that their imaging approach could be applied to other point scanning-based microscopy and imaging approaches in which it is important to image with low levels of light.

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Materials provided by The Optical Society. Note: Content may be edited for style and length.

Results published today show it is possible to identify Parkinson’s based on compounds found on the surface of skin. The findings offer hope that a pioneering new test could be developed to diagnose the degenerative condition through a simple and painless skin swab.
Scientists at The University of Manchester have developed a technique which works by analysing compounds found in sebum — the oily substance that coats and protects the skin — and identifying changes in people with Parkinson’s Disease. Sebum is rich in lipid-like molecules and is one of the lesser studied biological fluids in the diagnosis of the condition. People with Parkinson’s may produce more sebum than normal — a condition known as seborrhoea.
The research has been funded by charities Parkinson’s UK and the Michael J. Fox Foundation as well as The University of Manchester Innovation Factory. The work was originally funded following an observation by Joy Milne, whose husband was diagnosed with Parkinson’s at the age of 45. Working with Dr Tilo Kunath at the University of Edinburgh, Joy demonstrated an incredible ability to distinguish a distinctive Parkinson’s odour in individuals using her sense of smell, even before symptoms emerge in those affected.
The team, led by Professor Perdita Barran, The University of Manchester, and the clinical lead Professor Monty Silverdale at Salford Royal Foundation Trust, recruited 500 people with and without Parkinson’s. Samples of sebum were taken from their upper backs for analysis. Using different mass spectrometry methods, 10 chemical compounds in sebum were identified which are elevated or reduced in people with Parkinson’s. This allows scientists to distinguish people with Parkinson’s with 85 per cent accuracy.
The team confirmed their earlier findings published in ACS Central Science that the volatile compounds on skin can be used to diagnose the condition, increasing the number of people sampled and including participants from the Netherlands, as well as the UK.
In a new study published today in Nature Communications, high resolution mass spectrometry was used to profile the complex chemical signature in sebum of people with Parkinson’s and show subtle but fundamental changes as the condition progresses. Detailed analysis showed changes in people with Parkinson’s in lipid (fat) processing and mitochondria. Problems with mitochondria — the tiny energy-producing batteries that power cells — are one of the hallmarks of Parkinson’s.

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This means this ‘world first’ testing strategy is not only useful in diagnosing Parkinson’s but also in monitoring the development of the condition. The skin swab could provide an incredibly important new tool in clinical trials helping researchers measure whether new, experimental treatments are able to slow, stop or reverse the progression of Parkinson’s.
The study unveiled novel diagnostic sebum-based biomarkers for Parkinson’s, provides insight into understanding of how the condition develops, and links lipid dysregulation to altered mitochondrial function.
These promising results published today could lead to a definitive test to diagnose Parkinson’s accurately, speedily and cost effectively. The team is now seeking funding to further develop the test and explore the potential for using the test to ‘stratify’ patients.
Working with the University of Manchester Innovation Factory, the team has patents filed for their diagnostic techniques and are planning to create a spin-out company to commercialise the new tests. They are also working to use this approach to develop tests for COVID-19 as shown in research last week in EClinical Medicine as well as other conditions and are actively seeking investors interested in supporting the drive to bring this technology to market.
Professor Perdita Barran, Professor of Mass Spectrometry at The University of Manchester, said: “We believe that our results are an extremely encouraging step towards tests that could be used to help diagnose and monitor Parkinson’s.
“Not only is the test quick, simple and painless but it should also be extremely cost-effective because it uses existing technology that is already widely available.
“We are now looking to take our findings forwards to refine the test to improve accuracy even further and to take steps towards making this a test that can be used in the NHS and to develop more precise diagnostics and better treatment for this debilitating condition.”
Parkinson’s tends to develop gradually and it may be many months, even years, before the symptoms become obvious enough for an individual to visit their GP. A DaTscan is regularly used to help specialists confirm the loss of dopamine-producing cells that cause the development of Parkinson’s. However, similar loss may also occur in some other rarer neurological conditions. With no molecular test for the condition, diagnosis is made by a neurologist based on a combination of symptoms such as tremor, slowness, stiffness and balance issues. However, many of the symptoms of Parkinson’s can overlap with other conditions, especially in the early stages when progression is gradual and symptoms are more subtle.
In a recent survey of more than 2,000 people with Parkinson’s carried out by Parkinson’s UK, more than a quarter (26 per cent) reported they were misdiagnosed with a different condition before receiving the correct Parkinson’s diagnosis.¹

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