Study for New LD Test Underway in Virginia

Medical topics with questions, information and discussion related to Lyme disease and other tick-borne diseases.
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Re: Study for New LD Test Underway in Virginia

Post by Margherita » Sun 26 May 2013 11:07

Source: Elsevier, volume 32, issue 4- Feb. 2011


The use of hydrogel microparticles to sequester and concentrate bacterial antigens in a urine test for Lyme disease

a) Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Boulevard, Manassas, VA 20110, USA
b) Department of Urology, S. Giovanni Bosco Hospital, Torino 10154, Italy
c) Department of Medicine and Experimental Oncology, University of Turin, Turin 10126, Italy
d) Ceres Nanosciences, LLLP, Manassas, VA 20110, USA
e) Department of Analytical Chemistry, Stockholm University, 106 91 Stockholm, Sweden

Hydrogel biomarker capturing microparticles were evaluated as a biomaterial to amplify the sensitivity of urine testing for infectious disease proteins. Lyme disease is a bacterial infection transmitted by ticks. Early diagnosis and prompt treatment of Lyme disease reduces complications including arthritis and cardiac involvement. While a urine test is highly desirable for Lyme disease screening, this has been difficult to accomplish because the antigen is present at extremely low concentrations, below the detection limit of clinical immunoassays. N-isopropylacrylamide (NIPAm) – acrylic acid (AAc) microparticles were covalently functionalized with amine containing dyes via amidation of carboxylic groups present in the microparticles. The dyes act as affinity baits towards protein analytes in solution. NIPAm/AAc microparticles functionalized with acid black 48 (AB48) mixed with human urine, achieved close to one hundred percent capture and 100 percent extraction yield of the target antigen. In urine, microparticles sequestered and concentrated Lyme disease antigens 100 fold, compared to the absence of microparticles, achieving an immunoassay detection sensitivity of 700 pg/mL in 10 mL urine. Antigen present in a single infected tick could be readily detected following microparticle sequestration. Hydrogel microparticles functionalized with high affinity baits can dramatically increase the sensitivity of urinary antigen tests for infectious diseases such as Lyme disease. These findings justify controlled clinical studies evaluating the sensitivity and precision of Lyme antigen testing in urine. ... 1210012895

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Re: Study for New LD Test Underway in Virginia

Post by Lorima » Sun 26 May 2013 13:27

Thanks Marguerita!

Full text is here:
"I have to understand the world, you see."
Richard Feynman

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Re: Study for New LD Test Underway in Virginia

Post by dlf » Mon 27 May 2013 3:08

Last night a friend sent this to me...........This has just been announced -
The Ceres Solution
The Ceres' Nanotrap® High Sensitivity Lyme Antigen Test uses the Nanotrap® technology to directly measure Lyme antigens in urine using a Western Blot format. The Nanotrap® test is designed to provide high sensitivity and accuracy, delivering confident results at the earliest stages of infection. The Nanotrap® Lyme test is currently undergoing clinical trials. Anticipated clincial availabilty in late 2013 and early 2014.
It will be fascinating to see the results of their clinical trials! It would appear that with the nano-technology they are using, they may have worked out some of the issues with urine antigen testing, however I suspect there may be a few things that could still make this a less than sensitive test than might be advertised.

This is an article from 2005 outlining many of the problems faced when developing urine antigen testing and which at the time resulted in only one of twelve acute stage erythema migrans confirmed patients testing positive.
Critical Evaluation of Urine-Based PCR Assay for Diagnosis of Lyme Borreliosis
Carolin Rauter,1 Markus Mueller,1 Isabel Diterich,1 Sabine Zeller,1 Dieter Hassler,2
Thomas Meergans,1 and Thomas Hartung1,*

The kidney plays a role in the clearance of plasma DNA, and the human kidney barrier is permeable to DNA molecules
In an experiment with laboratory mice, only 0.06% of subcutaneously injected DNA was excreted into the urine within 3 days in a polymeric form. Thus, it appears unlikely that sufficient Borrelia DNA for a positive PCR result would be contained in 10 ml of urine.
Addition of EDTA to the samples immediately after collection inhibits nuclease activity in human urine, but DNA destruction due to nuclease activity in the bladder could not be ruled out. The predominant fraction of DNA fragments found in human urine was 150 bp to 250 bp. This is in line with our results, where the nuclease activity on plasmid DNA did not lead to complete degradation. Since the amplicon of our standard PCR is about 350 bp in length, most of the DNA fragments would be too short for amplification. But it could be that Borrelia DNA is bound to proteins and may thus be protected from nucleases better than the naked DNA used in our experiments. If urine of LB patients did contain Borrelia DNA, improved detection by inhibition of nuclease activity already in the bladder might be attained by systematic adjustment of the pH in the urine to at least pH 8 by bicarbonate-mediated alkalization of urine in vivo.
Taken together, we have developed an optimized, well-controlled, and highly sensitive PCR protocol for the species-specific detection of Borrelia DNA. Extrapolations indicate that even with this protocol, it is unlikely that Borrelia DNA will be detected in patient urine. In line, we were only able to detect Borrelia DNA in the urine of 1 of 12 EM patients. Although only 12 patient samples were tested, the combined results of spiked and patient urine samples enforce doubts that urine is a suitable material for the diagnosis of LB.

I wonder if they will find that the issues posed by urine having to first pass through a person's kidneys and bladder will be a problem with the trial.

Here is another article written back in the dark ages of such testing development, but again they seem to make a valid point. ... 37/?page=1

Infect Immun. 1991 January; 59(1): 269–278.
PMCID: PMC257737
Molecular detection of persistent Borrelia burgdorferi in the urine of patients with active Lyme disease.
J L Goodman, P Jurkovich, J M Kramber, and R C Johnson

In the Discussion section, the results of testing done by others which they mentioned seemed to point to sensitivity issues for urine antigen testing based on strain variation and also because of alterations in OspA.

This also makes me wonder whether the ticks that were tested in the article posted by Lorima and which were reported as either positive or negative were also tested with another form of more conventional PCR and whether all the negatives were true negatives, or only negative by their testing and were possibly different strains from what they used, or had somewhat varied OspA. Clearly that could pose sensitivity problems and would have to be addressed for use with a wider patient audience. Also, I imagine they would have to test against patients who had been vaccinated, not just one dog, to see if it doesn't react positively to the newer vaccine being tested in Europe.

I guess we will find out in due time.

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Re: Study for New LD Test Underway in Virginia

Post by Camp Other » Mon 27 May 2013 3:14

Lorima wrote:Thanks Marguerita!

Full text is here:
Oh cool - thank you both.

Do you know the first author on this publication was 18 years old when she came up with the idea for this test?

I wrote about her back in 2011. She won an Intel award.

You can watch a video of her here and read about her patent: ... tests.html

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Re: Study for New LD Test Underway in Virginia

Post by RitaA » Fri 25 Sep 2015 22:45

From an article posted in 2012:
The inspiration for the test started about two and a half years ago when a high school student from Lucketts, Virginia, joined Mason’s Aspiring Scientists Summer Internship Program and worked with Luchini and Liotta. Temple Douglas, now a junior at Princeton University, had family members who suffered from Lyme disease.
Here's an update on what Temple Douglas has been up to since then. What a remarkable young woman! ... ancer.html
Biomedical engineering student develops new Lyme disease test, now working in cancer detection

September 25, 2015 by Steven Mackay

Temple Douglas of Lucketts, Virginia, is one of several doctoral students working in a Virginia Tech microfluidics lab helping discover new cancer detection methods. The task is well suited for her. While in high school, Douglas developed a new test for Lyme disease that uses urine.

Douglas works in the university's BioTrans Interdisciplinary Graduate Education Program, part of the Virginia Tech Department of Biomedical Engineering and Mechanics.

Previously, she earned a bachelor's in physics from Princeton in 2014, with certificates in materials science and engineering, and biophysics. Before she was at Princeton, though, Douglas already had cut her teeth in the biomedical field. Before her senior year at Thomas Jefferson High School for Science and Technology, Douglas was working at nearby George Mason University through its Aspiring Scientists Summer Internship Program.

That was in 2010. Several members of her family had recently contracted Lyme disease – an infectious bacterial disease transmitted via a tick bite – and Douglas became aware of limits in current blood-based tests that relies on the detection of antibodies, which can take weeks and still be unreliable.

"Antibodies form weeks after the tick bite, after which you have lost a window of time when antibiotics could have been started," said Douglas. "Antibody reactions can be incomplete, often leading to false positives or false negatives. By detecting bacterial protein rather than the patient's antibody response, it is possible to know earlier and more accurately whether someone has contracted Lyme disease."

Douglas' mentor at George Mason, Alessandra Luchini, had developed nanoparticles to use for the concentration and detection of low molecular weight proteins and biomarkers. Temple thought the same nanoparticles could detect Borrelia burgdorferi antigen fragments – that's the bacteria that forms Lyme disease—in urine, allowing for a more sensitive diagnostic test. George Mason allowed Temple to work on the method for her high school senior research project.

After graduation, before Princeton, Douglas continued working on the Lyme disease test in the summer and it went into clinical testing soon afterward. George Mason researchers carried the tests onward, and announced this past spring tests in early-stage Lyme disease patients had a near perfect detection rate.

The test is now commercially available and being used in some hospitals in Northern Virginia.

The timing is fortunate. Virginia and several other Southern states have reported increases in Lyme disease cases. The disease can be brutal, moving from early stage headaches and fatigue to achy joints, heart and central nervous system issues, and stroke, according to the Centers for Disease Control.

Temple now works with biomedical engineering Professor Rafael Davalos and postdoctoral associate Jaka Cemazar on microfluidics in Kelly Hall, where the Institute for Critical Technology and Applied Science is based. The goal is to detect tumor-initiating cells, also known as cancer stem cells, using a microfluidic platform to separate cancer stem cells or tumor-initiating cells that form new tumors, based on their electrical properties. According to the lab, this method could be used for personalized cancer treatment to develop drugs to target the most aggressive cells.

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Re: Study for New LD Test Underway in Virginia

Post by ChronicLyme19 » Sat 26 Sep 2015 15:17

We still don't know the sensitivity of this test yet do we?
announced this past spring tests in early-stage Lyme disease patients had a near perfect detection rate.
The test is now commercially available and being used in some hospitals in Northern Virginia.
A near perfect detection rate compared to what? If it's 2T testing, that's not good enough.

I do agree this is the right strategy for a quick test. Advances in nanotechnology should be able to increase the sensitivity of testing greatly, and look for the bacteria directly, rather than an immune response. This in conjunction with a gold standard culture to truly verify the detection rate would be awesome.

Edited to add:
I do have to say I'm biased to these types of methods being as my background is in materials science and engineering. :)
Half of what you are taught is incorrect, but which half? What if there's another half missing?

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