Nanotrap measurement of OspA urinary shedding may lead to improved testing

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
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dlf
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Nanotrap measurement of OspA urinary shedding may lead to improved testing

Post by dlf » Sat 7 Nov 2015 0:11

Some results regarding the Ceres Nanotrap technology; hopefully we will see a full-text version eventually.

http://www.ncbi.nlm.nih.gov/pubmed/26537892
J Transl Med. 2015 Nov 4;13(1):346. doi: 10.1186/s12967-015-0701-z.
Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis.
Magni R1,2, Espina BH3, Shah K4, Lepene B5, Mayuga C6, Douglas TA7, Espina V8, Rucker S9, Dunlap R10, Petricoin EF11, Kilavos MF12, Poretz DM13, Irwin GR14, Shor SM15, Liotta LA16, Luchini A17.

Abstract
OBJECTIVES:
Prompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB.
METHOD:
We employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation.
RESULTS:
OspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7-30 pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p < 10(-6)). For 10 patients who exhibited persistence of the EM rash during the course of antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e(-15)). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein.
CONCLUSIONS:
OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative.


dlf
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Re: Nanotrap measurement of OspA urinary shedding may lead to improved testing

Post by dlf » Sat 7 Nov 2015 15:29

Many thanks for posting the link nnecker!

I am very impressed by the quality of this study and the potential for this technology to be both ground breaking and game changing for detecting active Lyme infection in patients (even after antibiotic treatment). Obviously more work needs to be done, but this technology shows real promise for becoming a clinically important diagnostic tool. :D

nnecker
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Re: Nanotrap measurement of OspA urinary shedding may lead to improved testing

Post by nnecker » Sat 7 Nov 2015 16:07

From the study:
Treated patients under clinical evaluation for persistent or recurrent LB.

Urinary OspA shedding was further evaluated in a cohort of 100 patients in a Lyme endemic geographic region who were under clinical surveillance for persistent or recurrent LB. All of these patients had been previously treated with antibiotics, and all patients had been followed because of prolonged chronic functional symptoms such as arthralgias, neurocognitive symptoms, and fatigue.

After the urine OspA scoring was completed, the clinical data was unblinded. For this special group of previously treated patients under surveillance for persistent or recurrent LB, 41/100 were positive for urinary OspA C-terminal peptides
If this is an accurate test,then the data is telling us that 59 % of patients with prolonged chronic functional symptoms are needlessly being given long term antibiotic treatment.

For the 41% who were positive after treatment,is it that this test is detecting spirochete debris?

duncan
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Re: Nanotrap measurement of OspA urinary shedding may lead to improved testing

Post by duncan » Sat 7 Nov 2015 16:36

This is a coup for early testing.

Moreover, this test seems to be making headway in the more difficult to gauge late stage post-treatment patients. (I am assuming that, by definition, each of those 41 post-treatment symptomatic patients who tested positive were late stage by the time of the application of this test.)

It appears that between a third and half of patients that have continued to proclaim their symptoms as unresolved following IDSA-recommended treatments - and told they could not still be infected - in fact still may be infected with Bb.

We are making progress.

ETA: Do we know of those 100 with persistent symptoms how many were treated during early stage vs late stage? Could the 41% be those that were treated early stage, and hence evidence of Bb through this new test more readily available, comparatively? Could the other 59% be those who received treatment in a later stage? I did not see that in the study, so speculation seems counter-productive, I suppose.

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ChronicLyme19
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Re: Nanotrap measurement of OspA urinary shedding may lead to improved testing

Post by ChronicLyme19 » Mon 9 Nov 2015 2:25

“The anti-OspA mAb epitope is conserved in common pathogenic species of Borrelia
The antigenic epitope OspA236-239 is highly conserved among major pathogenic Borrelia strains, which include: Borrelia burgdorferi sensu stricto, several Borrelia burgdorferi sensu lato (Borrelia garinii, Borrelia valaisiana, Borrelia bissettii, Borrelia afzelii, and Borrelia spielmanii) and additional more recently characterized pathogenic species (Additional file 1: Table S2). BLAST analysis [32] performed on the sequence of the fragment OspA219-253 KTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGT identified by mass spectrometry showed that it was not homologous to any human protein and not homologous to any other non-Borrelia spirochetes (Additional file 1: Table S2).”
The table does not mention borrelia miyamotoi if anyone was wondering.
“OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative.”
This is really promising if it holds true as it may have the ability to determine if persisting symptoms are from ongoing infection or not without having to look at a patients immune response. OspA and OspC I think were the two bands that showed up most frequently in the IGeneX tests for those with PTLDS. I know when I’m on antibiotics, my symptoms disappear and then when I stop antibiotics they come back with a vengeance. So I’m wondering would someone like me only test positive when I’m off antibiotics and am symptomatic or would I test positive either way or not at all? How would the round body persisters affect this test? It’s been shown to be metabolically dormant, so does it still produce the OspA?
“For this special group of previously treated patients under surveillance for persistent or recurrent LB, 41/100 were positive for urinary OspA C-terminal peptides (Additional file 1: Table S6).”
It’s not clear to me of these 100 patients if they were still on antibiotics or not at the time the test was performed, only that they had at least one round of Lyme antibiotics in the past.

Interesting bits…
“Positive OspA bands are normally visible in the 28–30 kDa range although lower molecular bands can be detected and successfully competed suggesting the presence of smaller OspA C-terminal domain protein fragments in urine. In some patients a high molecular weight band at ~60 kDa was detected and this was determined to be a dimer of OspA by mass spectrometry sequencing (Additional file 1: Figure S1).”
“Most, if not all, of these patients have a negative Lyme serology by the 2 tier criteria. Thus our findings of 41 % positive patients in this population cannot be defined as a level of sensitivity and specificity, since none, or all, of these patients could actually have had an active Borrelia infection. Importantly, our data provides the first antigenic evidence that at least 41 % of these patients may have an active Bb infection. Therefore these data contribute significant new information to the debate about chronic Lyme disease.”
The awesome thing about this is that it wasn’t 100% of the PTLDS subjects. Meaning that if there is a faction of us with ongoing infection, maybe it isn’t all. Some people seem to benefit from extended antibiotic therapy while others do not, and this certainly could help explain the divide. One step closer to figuring out who needs retreatment and what type. :woohoo:
Half of what you are taught is incorrect, but which half? What if there's another half missing?

Lorima
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Re: Nanotrap measurement of OspA urinary shedding may lead to improved testing

Post by Lorima » Mon 9 Nov 2015 14:01

"Importantly, our data provides the first antigenic evidence that at least 41 % of these patients may have an active Bb infection."
(My emphasis)

"The awesome thing about this is that it wasn’t 100% of the PTLDS subjects. Meaning that if there is a faction of us with ongoing infection, maybe it isn’t all."

I wouldn't assume that all patients with living Borrelia in their bodies would be shedding OspA ( or any antigen) into their urine at all times. For example, moderate numbers of Borrelia in the brain might not be expected to shed antigen continuously into the blood stream and hence into urine.

But if it can be shown repeatedly that this assay is virtually 100% specific, it should encourage treatment of those patients who are positive on it.

Now we just need to revive research into treatment protocols for late-stage LD. That got frozen in the late 1980s by the fiction that treatments then current were virtually 100% effective, so that no new approach was needed or justified.
"I have to understand the world, you see."
Richard Feynman

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Re: Nanotrap measurement of OspA urinary shedding may lead to improved testing

Post by X-member » Mon 9 Nov 2015 16:08

A quote from an earlier post:
OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis)....
Interesting! 8-)

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Re: Nanotrap measurement of OspA urinary shedding may lead to improved testing

Post by X-member » Thu 12 Nov 2015 5:58

Perhaps this belong in this thread?

Dr Marie Kroun, Denmark give the following information (regarding the study in the first post in this thread) on facebook:

Two quotes:
Det er ikke alle Borrelia der udtrykker OspA.
- og f.eks. Borrelia miyamotoi udtrykker slet ikke OspA
- dvs. den test metode kan teoretisk næppe pålideligt påvise alle Borrelia infektioner!
Translation with google translate (and partly by me):
It is not all Borrelia that express OspA.
- and, for example. Borrelia miyamotoi do not express OspA
- i.e., the test method can theoretically hardly reliably detect all Borrelia infections!
https://www.facebook.com/daninfekt/?fref=nf

Dr Marie Kroun also posted the links (on facebook) that I posted in the thread below:

OspA causes immune suppression, Part I

http://www.lymeneteurope.org/forum/view ... 9&start=10

I don't know if I have posted the links in the right place in this forum.

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ChronicLyme19
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Re: Nanotrap measurement of OspA urinary shedding may lead to improved testing

Post by ChronicLyme19 » Tue 17 Nov 2015 2:38

- and, for example. Borrelia miyamotoi do not express OspA
- i.e., the test method can theoretically hardly reliably detect all Borrelia infections!
That was one of my concerns...
Half of what you are taught is incorrect, but which half? What if there's another half missing?

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