Lyme disease detection weeks sooner than current tests

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Rutgers researcher leads team analyzing more exact methods to diagnose the most common tick-borne infection

Researchers have developed techniques to detect Lyme disease bacteria weeks sooner than current tests, allowing patients to start treatment earlier.

The findings appear in the journal Clinical Infectious Diseases. The authors include scientists from Rutgers Biomedical and Health Sciences, Harvard University, Yale University, the National Institute of Allergy and Infectious Diseases, FDA, Centers for Disease Control and Prevention, and other institutions.

The new techniques can detect an active infection with the Lyme bacteria faster than the three weeks it takes for the current indirect antibody-based tests, which have been a standard since 1994. Another advantage of the new tests is that a positive result in blood indicates the infection is active and should be treated immediately, allowing quicker treatment to prevent long-term health problems. The techniques detect DNA or protein from the Lyme disease bacteria Borrelia burgdorferi.

“These direct tests are needed because you can get Lyme disease more than once, features are often nondiagnostic and the current standard FDA-approved tests cannot distinguish an active, ongoing infection from a past cured one,” said lead author Steven Schutzer, a physician-scientist at Rutgers New Jersey Medical School. “The problem is worsening because Lyme disease has increased in numbers to 300,000 per year in the United States and is spreading across the country and world.”

Lyme disease signs frequently, but not always, include a red ring or bull’s eye skin rash. When there is no rash, a reliable laboratory test is needed and preferably one that indicates active disease. The only FDA-approved Lyme disease tests rely on detecting antibodies that the body’s immune system makes in response to the disease. Such a single antibody test is not an active disease indicator but rather only an exposure indicator — past or present.

“The new tests that directly detect the Lyme agent’s DNA are more exact and are not susceptible to the same false-positive results and uncertainties associated with current FDA-approved indirect tests,” said Schutzer. “It will not be surprising to see direct tests for Lyme disease join the growing list of FDA-approved direct tests for other bacterial, fungal and viral infections that include Staphylococcus, Streptococcus, Candida, influenza, HIV, herpes and hepatitis, among others.”

Learn more: New Techniques Can Detect Lyme Disease Weeks Before Current Tests

 

 

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Major implications for the treatment of tick-borne diseases like Lyme disease

Ixodes scapularis ticks transmit the pathogens of Lyme disease, resulting a multisystem illness in a variety of animals and humans. The image shows bottom side a live Ixodes tick as seen under a confocal immunofluorescence microscope.
CREDIT
Dr. Utpal Pal, University of Maryland

Findings have major implications for tick-borne diseases like Lyme disease

A University of Maryland (UMD) researcher has uncovered a mechanism by which the bacteria that causes Lyme disease persists in the body and fights your early, innate immune responses. Dr. Utpal Pal, Professor in Veterinary Medicine, has been studying the Borrelia burgdorferi bacteria throughout his twelve years with UMD, and his work has already produced the protein marker used to identify this bacterial infection in the body. Now, Dr. Pal has isolated a protein produced by the bacteria that disables one of the body’s first immune responses, giving insight into mechanisms that are largely not understood. He has also observed a never-before-seen phenomena demonstrating that even without this protein and with the immune system responding perfectly, the bacteria can spring back in the body weeks later. Understanding this bacteria, which is amongst only a few pathogens that can actually persist in the body for long periods of time, has major implications for the treatment of tick-borne diseases like Lyme disease, which is an increasingly chronic and consistently prevalent public health issue.

“Most people don’t realize that they actually are walking around with more bacterial cells in their bodies than their own cells, so we are really bags of bacteria,” explains Pal. “Most are good, but the second your body detects something that is a pathogen and can cause disease, your immune system starts to work.” The body sends a first, nonspecific wave of attack to kill the bacteria detected that doesn’t belong. This happens within a few hours to days. If this doesn’t work, it takes seven to ten days to learn about the enemy and send a large second wave of reinforcements to kill what is left. “Lyme disease is actually caused by your immune system,” explains Pal. “This bacteria wins the first battle, and your body overreacts so much that it causes intense inflammation in all the joints and areas that the bacteria spreads by sending so many reinforcements to kill it. Borrelia is then killed, but the inflammation remains and causes many of your symptoms for Lyme disease. That is why killing Borrelia in the first wave of immunity is so important.”

The Centers for Disease Control and Prevention (CDC) estimate about 300,000 cases of Lyme disease annually in the United States. However, these cases are largely underestimated and reported due to the attention given to mosquito-transmitted diseases like malaria. “The majority of all vector-borne diseases in the US are actually tick-borne, and 6 of the 15 distinct tick diseases are transmitted by the Ixodes tick we study in our lab,” says Pal. “The symptoms of these diseases present similarly to many other illnesses and are hard to pin down, so they are vastly underreported and an even bigger public health concern locally and globally than people realize.” Now, chronic Lyme disease is a growing concern. Six to twelve months after traditional antibiotic therapy, many people have non-objective symptoms that return with varying intensity and no current treatment strategy, known as Post-Treatment Lyme Disease Syndrome.

Dr. Pal’s research has shed some light on this issue and paved the way for future research and treatment options by discovering that even without the protein used to beat the first wave of immune defense, infection can reoccur in the body weeks later. “This means there is a second line of defense for Borrelia just like for our body’s immune system. This had never been observed before and gives us insight into what could be causing these chronic Lyme disease cases,” explains Pal.

Dr. Pal is frequently consulted for his expertise and has written books on this highly versatile bacteria. The federal government has recently put more emphasis on tick-borne disease research and a major public health issue with the passage of the 21st Century Cures Act. As part of this, Dr. Pal was asked to serve on a Tick-Borne Disease Working Group Subcommittee for the U.S. Department of Health & Human Services (DHHS) focused on vaccines and therapeutics for tick-borne diseases, driving future research in the field. Dr. Pal currently holds two concurrent multi-million dollar RO1 grants from the National Institutes of Health (NIH) for this work, only granted for highly important and influential research. “I am fascinated by Borrelia, and this discovery will open the door for much more work to treat and control important diseases like Lyme disease,” says Pal.

Learn more: UMD researcher uncovers protein used to outsmart the human immune system

 

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Faster test results for HIV, Lyme disease, syphilis, rotavirus and other infectious conditions

Assistant Professor Shawn Putnam of UCF’s College of Engineering & Computer Science.

A UCF researcher has combined cutting-edge nanoscience with a magnetic phenomenon discovered more than 170 years ago to create a method for speedy medical tests.

The discovery, if commercialized, could lead to faster test results for HIV, Lyme disease, syphilis, rotavirus and other infectious conditions.

“I see no reason why a variation of this technique couldn’t be in every hospital throughout the world,” said Shawn Putnam, an assistant professor in the University of Central Florida’s College of Engineering & Computer Science.

At the core of the research recently published in the academic journal Smallare nanoparticles – tiny particles that are one-billionth of a meter. Putnam’s team coated nanoparticles with the antibody to BSA, or bovine serum albumin, which is commonly used as the basis of a variety of diagnostic tests.

By mixing the nanoparticles in a test solution – such as one used for a blood test – the BSA proteins preferentially bind with the antibodies that coat the nanoparticles, like a lock and key.

That reaction was already well known. But Putnam’s team came up with a novel way of measuring the quantity of proteins present. He used nanoparticles with an iron core and applied a magnetic field to the solution, causing the particles to align in a particular formation. As proteins bind to the antibody-coated particles, the rotation of the particles becomes sluggish, which is easy to detect with laser optics.

The interaction of a magnetic field and light is known as Faraday rotation, a principle discovered by scientist Michael Faraday in 1845. Putnam adapted it for biological use.

“It’s an old theory, but no one has actually applied this aspect of it,” he said.

Other antigens and their unique antibodies could be substituted for the BSA protein used in the research, allowing medical tests for a wide array of infectious diseases.

The proof of concept shows the method could be used to produce biochemical immunology test results in as little as 15 minutes, compared to several hours for ELISA, or enzyme-linked immunosorbent assay, which is currently a standard approach for biomolecule detection.

Learn more: New, Old Science Combine to Make Faster Medical Test

 

 

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Early-detection urine test for Lyme disease works

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After three years and 300 patients, George Mason University researchers have proof that their early-detection urine test for Lyme disease works.

It’s the largest study of its kind looking at early-stage indicators for Lyme disease, said Lance Liotta, co-director and medical director of the George Mason-based Center for Applied Proteomics and Molecular Medicine. “We are looking at a highly specific protein shed from the surface of the bacteria that causes Lyme.”

And now they’re applying the approach to Ebola, malaria and tuberculosis, among other diseases. The Mason team is working side by side with the private company Ceres Nanoscience, which Liotta and his co-director Chip Petricoin co-founded. A test that works like a pregnancy test could be used in undeveloped countries to quickly identify disease, even when patients aren’t near a hospital, he said.

The National Institutes of Health funded the research that led to Mason’s patented technology, which traps tell-tale clues (such as the Lyme bacteria protein) that a disease is present. The Mason technology, which is licensed to Ceres, works during the earliest stages of disease and finds the tiniest traces missed by most diagnostic tests.

In the case of Lyme disease, some patients may still have active cases but traditional tests don’t register it, Liotta said. These patients may not be receiving the additional round of treatment they need, he said.

“If the patient gets better, the test goes negative,” Liotta said. “It’s a good way to monitor the patient.”

“We’re looking to repeat the story again with these other diseases,” said Alessandra Luchini, a Mason professor who spearheaded the Lyme test research, is a co-inventor of the technology, and continues to develop new applications. “Other targets for the new type of test include Chagas disease, which is infectious and caused by a parasite, and toxoplasmosis, another parasite-borne disease.”

Learn more: Lyme disease is no match for Mason researchers

 

 

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New Experimental Test Detects Signs of Lyme Disease Near Time of Infection

Deer, or black-legged, ticks can transmit Lyme disease to humans during feeding, when they insert their mouth parts into the skin. A new experimental test developed at NIST has been shown to detect the disease near the time of infection, earlier than the standard blood test now used. Credit: James Gathany/CDC

Deer, or black-legged, ticks can transmit Lyme disease to humans during feeding, when they insert their mouth parts into the skin. A new experimental test developed at NIST has been shown to detect the disease near the time of infection, earlier than the standard blood test now used.
Credit: James Gathany/CDC

When it comes to early diagnosis of Lyme disease, the insidious tick-borne illness that afflicts about 300,000 Americans annually, finding the proverbial needle in the haystack might be a far easier challenge—until now, perhaps. An experimental method developed by federal and university researchers appears capable of detecting the stealthy culprit Lyme bacteria at the earliest time of infection, when currently available tests are often still negative.

The team suggests the approach might also be useful for early detection of other elusive bacterial infections. The collaborators—from the National Institute of Standards and Technology (NIST), Institute for Bioscience and Biotechnology Research, and Johns Hopkins School of Medicine—recently reported the successful first trial of their new method.

“Our hypothesis was that Lyme bacteria shed vesicle-like particles—or fragments—derived from the cell wall of the bacteria circulating in the serum of individuals. These particles would contain membrane proteins that can be detected to provide a unique indicator of infection,” explains NIST research chemist Larik Turko.

The challenge was to detect these bacterial membrane proteins among the far, far more plentiful proteins normally present in serum, the watery, cell-free component of blood. The researchers speculated that running serum samples through a high-speed centrifuge—a standard step in chemistry labs—might selectively concentrate the larger, heavier fragments containing the bacterial membrane proteins into pellets. In effect, they predicted, this step would separate the wheat—the sparse target proteins—from the chaff—the much more abundant human serum proteins.

The new method’s promise was demonstrated in tests on serum samples drawn from three patients with undetected Lyme disease at the time of their initial doctor visit. By customizing standard analytical techniques for determining the types and amounts of chemicals in a sample, the team detected extremely small amounts of the target protein in all three samples.

For chemistry buffs, the protein in enriched samples was present at a level of about four billionths of a millionth of a mole, the standard unit for amount of substance.

In one patient, the experimental method detected the bacteria three weeks before infection was confirmed with the standard blood tests now used. For the other two, infection was detected simultaneously by the two methods.

“The complexity of Lyme disease, combined with lack of biomarkers to measure infection, has slowed progress,” study collaborator John Aucott, head of the Johns Hopkins Lyme Disease Clinical Research Center, said in advance of a session on precision and personalized medicine this weekend at the AAAS 2016 Annual Meeting in Washington, D.C. “Now, thanks to recent advances in technology, the tiniest concentration of blood molecules can now be detected, molecules that were previously ‘invisible’ to scientists.”

The current standard blood test for Lyme disease exposes the infection only after antibodies have accumulated to detectable levels, which can take up to 4 to 6 weeks. If patients exhibit a telltale bull’s-eye rash, diagnosis and treatment can begin earlier. But the rash does not occur in 20 to 30 percent of Lyme disease patients, according to the Centers for Disease Control and Prevention.

Rather than waiting for an infected person’s immune system to produce noticeable amounts of antibodies, the team chose to home in on the bacteria itself—specifically, proteins the bug sheds when attacked by the body’s defenses.

“From many candidates, we chose one that is both easily distinguished from human serum proteins and an unambiguous indicator of the bacteria,” Turko says. “This protein, which resides on the outer surface of membranes, became the target of our search in serum samples.”

But finding that target required an important preliminary step to ensure the accuracy of their measurements: making a reference sample that contained ample amounts of the target protein. With the reference sample, the team established the unmistakable signature the bug’s outer-surface membrane protein would yield when they examined samples drawn from patients. As a result of these steps, the team could detect the copies of the target protein, even though human proteins were 10 million times more plentiful.

“We believe that this approach may be universally applicable to detection of other bacterial infections in humans,” the researchers write.

Learn more: New Experimental Test Detects Signs of Lyme Disease Near Time of Infection

 

 

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