Xenodiagnosis in Humans to Detect Bb infection

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
RitaA
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Xenodiagnosis in Humans to Detect Bb infection

Post by RitaA » Fri 14 Feb 2014 7:21

http://cid.oxfordjournals.org/content/e ... 9.abstract
Clin Infect Dis. (2014) doi: 10.1093/cid/cit939 First published online: February 11, 2014

Xenodiagnosis to Detect Borrelia burgdorferi Infection: A First-in-Human Study

Adriana Marques 1,
Sam R. Telford III 2,
Siu-Ping Turk 1,
Erin Chung 3,
Carla Williams 4,
Kenneth Dardick 5,
Peter J. Krause 6,
Christina Brandeburg 3,
Christopher D. Crowder 7,
Heather E. Carolan 7,
Mark W. Eshoo 7,
Pamela A. Shaw 8, and
Linden T. Hu 3

- Author Affiliations

1 Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
2 Department of Infectious Disease and Global Health, Cummings School of Veterinary Medicine, Tufts University
3 Department of Medicine, Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, Massachusetts
4 SAIC-Frederick, Inc, NCI-Frederick, Frederick, Maryland
5 Mansfield Family Practice, Storrs
6 Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Yale School of Medicine, New Haven, Connecticut
7 Ibis Biosciences, Inc., a subsidiary of Abbott Company, Carlsbad, California;
8 Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

Correspondence: Linden Hu, MD, Tufts Medical Center, 800 Washington St, Box 41, Boston, MA 02111 (lhu@tuftsmedicalcenter.org).

Presented in part: 2013 International Conference on Lyme Borreliosis and other Tick Borne Diseases, Boston, Massachusetts, 18–21 August 2013. Poster B040.

Abstract

Background.

Animal studies suggest that Borrelia burgdorferi, the agent of Lyme disease, may persist after antibiotic therapy and can be detected by various means including xenodiagnosis using the natural tick vector (Ixodes scapularis). No convincing evidence exists for the persistence of viable spirochetes after recommended courses of antibiotic therapy in humans. We determined the safety of using I. scapularis larvae for the xenodiagnosis of B. burgdorferi infection in humans.

Methods.

Laboratory-reared larval I. scapularis ticks were placed on 36 subjects and allowed to feed to repletion. Ticks were tested for B. burgdorferi by polymerase chain reaction (PCR), culture, and/or isothermal amplification followed by PCR and electrospray ionization mass spectroscopy. In addition, attempts were made to infect immunodeficient mice by tick bite or inoculation of tick contents. Xenodiagnosis was repeated in 7 individuals.

Results.

Xenodiagnosis was well tolerated with no severe adverse events. The most common adverse event was mild itching at the tick attachment site. Xenodiagnosis was negative in 16 patients with posttreatment Lyme disease syndrome (PTLDS) and/or high C6 antibody levels and in 5 patients after completing antibiotic therapy for erythema migrans. Xenodiagnosis was positive for B. burgdorferi DNA in a patient with erythema migrans early during therapy and in a patient with PTLDS. There is insufficient evidence, however, to conclude that viable spirochetes were present in either patient.

Conclusions.

Xenodiagnosis using Ixodes scapularis larvae was safe and well tolerated. Further studies are needed to determine the sensitivity of xenodiagnosis in patients with Lyme disease and the significance of a positive result.

Clinical Trials Registration. NCT01143558.
http://cid.oxfordjournals.org/content/e ... 42.extract
Clin Infect Dis. (2014) doi: 10.1093/cid/cit942 First published online: February 11, 2014

Xenodiagnosis for Posttreatment Lyme Disease Syndrome: Resolving the Conundrum or Adding to It?

Linda K. Bockenstedt 1 and
Justin D. Radolf 2

- Author Affiliations

1 Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut
2 Department of Medicine, University of Connecticut Health Center, Farmington

Correspondence: Linda K. Bockenstedt, MD, Section of Rheumatology, Yale University School of Medicine, PO Box 208031, New Haven, CT 06520-8031 (linda.bockenstedt@yale.edu).

The first reports of Lyme disease in Connecticut in the mid-1970s, followed by the discovery of its tick-borne etiologic agent, Borrelia burgdorferi, in 1983, has spawned one of the longest controversies in the history of biomedical research [1]. Whereas some clinical signs, such as the hallmark skin lesion erythema migrans (EM), can be explained by the inflammatory response elicited by spirochetes, other features remain enigmatic. Impressive generalized symptomatology can accompany EM and last for weeks to months following therapy and resolution of clinical signs. A minority of people (<10%) continue to experience fatigue, musculoskeletal pain, and/or cognitive dysfunction, a condition called posttreatment Lyme disease syndrome (PTLDS) [2]. The issue at the heart of the current, often acrimonious debate is whether persistent infection drives protracted symptomatology.

Four randomized, placebo-controlled trials have evaluated whether extended courses of antimicrobials ameliorate symptoms, ostensibly by eliminating persistent organisms [3–5]. The first 2 trials enrolled seropositive subjects with a previous episode of Lyme disease and seronegative subjects with physician-documented EM [3]. These were the most rigorous, as they evaluated therapeutic responses in a large number of subjects and also sought evidence of infection in blood and cerebrospinal fluid using culture and polymerase chain reaction (PCR). The treatment regimen (1 month of intravenous ceftriaxone followed by 2 months of oral doxycycline) was selected because both agents have good tissue penetration (including the central nervous system) and well-documented in vitro and in vivo activity against B. burgdorferi. The results of the intervention were clear: No evidence was obtained for persistent spirochetes, and antimicrobials provided no benefit over placebo. The remaining 2 trials showed either a similar lack of efficacy after 10 weeks of ceftriaxone [4 …
It's a shame the extract doesn't include anything about xenodiagnoses itself. I guess we'll have to rely on notes from someone with access to the full article to better understand the title. *

By the way, there's a thread from 2011 about xenodiagnosis for Lyme disease in case anyone is interested in that earlier discussion:

http://www.lymeneteurope.org/forum/view ... f=5&t=3387

Edited to add:

* The blogger Relative Risk has provided notes from the full article. I've posted them below (on page 2 of this thread).
Last edited by RitaA on Sat 15 Feb 2014 4:13, edited 1 time in total.

duncan
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Re: Xenodiagnosis in Humans to Detect Bb infection

Post by duncan » Fri 14 Feb 2014 16:34

It is good to see first hand how Science has evolved over the last 300 or 400 years, from leeching to ticking.

I can perhaps appreciate the potential of Xenodiagnosis in very early Lyme. But after it has disseminated - when it is widely acknowledged to be difficult to locate in blood...when many believe the spirochetes have retreated into sanctuaries like tissue and joints...Not sure how applicable the procedure would be.

I realize there are some who support this approach. Personally, it has the feel of a misdirection.

Martian
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Re: Xenodiagnosis in Humans to Detect Bb infection

Post by Martian » Fri 14 Feb 2014 16:55

duncan wrote:I can perhaps appreciate the potential of Xenodiagnosis in very early Lyme. But after it has disseminated - when it is widely acknowledged to be difficult to locate in blood...when many believe the spirochetes have retreated into sanctuaries like tissue and joints...Not sure how applicable the procedure would be.
Ticks naturally become infected with Bb when sucking blood. So apparently there are often Bb spirochetes in the blood of a host. However, I do find it surprising that this works so well, considering the little amount of blood a tick is consuming. Is Bb abundant in the blood of the host after all, or is there some other mechanism involved? Are Bb spirochetes somehow signalled that the host in having a tick bite and do they therefore enter the bloodstream?

duncan
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Re: Xenodiagnosis in Humans to Detect Bb infection

Post by duncan » Fri 14 Feb 2014 17:32

I'm not sure it has been demonstrated it works so well in humans.

How much is the NIH throwing at Lyme each year? Around $20 mill?

What percentage of that allotment is accounted for by this study?

Is this really the NIH showing the world that it is pursuing all possible avenues on diagnostic fronts??

X-member
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Re: Xenodiagnosis in Humans to Detect Bb infection

Post by X-member » Fri 14 Feb 2014 18:01

Is a disseminated infection (= borrelia bacteria also in the blood) common in cases that only had erythema migrans?

Edit to add:

And is borrelia bacteria really present in blood in cases that have no signs of an active infection (= PTLDS)?

http://www.lymeneteurope.org/forum/view ... sik#p26756

A quote (from the link above):
1. Spirochetes could be demonstrated in BLOOD by dark-field microscopy during all ACTIVE stages of pathogenesis!

X-member
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Re: Xenodiagnosis in Humans to Detect Bb infection

Post by X-member » Fri 14 Feb 2014 18:17

Maybe this belong in this thread too?

Searching for Persistence of Infection in Lyme Disease

http://clinicaltrials.gov/ct2/show/NCT0 ... 558&rank=1

A quote:
Eligibility:

Individuals at least 18 years of age who are in one of the following categories:

Have had the erythema migrans rash (a symptom of Lyme infection) and received antibiotic therapy less than 4 months ago (but have not had antibiotics in the previous month).

Have the erythema migrans rash and received less than 2 days of antibiotic therapy.

Have been diagnosed with early or late Lyme disease, have received antibiotic therapy, but still have high levels of antibodies against the bacteria at least 6 months after therapy, and have not received antibiotics in the past 3 months.

Have been diagnosed with early or late Lyme disease, have received antibiotic therapy, and have new complaints of fatigue or other symptoms that are persistent for at least 6 months after completion of antibiotic therapy, and have not received antibiotics in the past 3 months.

Have been diagnosed with Lyme arthritis and have not yet received antibiotic therapy.

Healthy volunteers who have not had Lyme disease will also be included in this study.

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LHCTom
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Re: Xenodiagnosis in Humans to Detect Bb infection

Post by LHCTom » Fri 14 Feb 2014 18:20

I suspect Adriana Marques is just going through the motions. She is on record saying persistence of the Borrelia spirochete does not occur. Asking her to study persistence is a bit like asking the hen to guard the hen house. There is nothing she would like better than to show Barthold et al are wrong. So she uses the same technique used in animal studies. There is a large difference between a mouse and humans which are dead end hosts. It might ruin her career and reputation if she accidentally found evidence but through careful study design, its easy to bias the results.

Xenodiagnosis is a form of culturing except it uses the tiny amount of blood the tick feeds upon. Then it uses the tick gut as a culture medium. It doesn't take a rocket scientist to understand that the number of spirochetes in the blood after dissemination and post immune response is VERY VERY low. This is why Wormser found he needed to use large amounts of blood just to culture acute Lyme while the spirochete load is still high. Eshoo who is involved also knows it takes lots of blood and multiple PCR tests per blood sample to achieve success. By increasing the amount of blood and then running multiple PCR tests, the odds of finding DNA goes up. So what does Adriana do? - she only tests one of 30 ticks. Doesn't the NIH have enough money to test ALL the ticks? I wonder how long she waited before running the PCR on the ONE of 30 ticks. Did she wait long enough for the spirochetes to multiply. I bet not.

Since the taxpayers paid for the study, why is the paper not posted on the NIH clinical trails site and why is the paper not free? Its should be a requirement of NIH funding that the papers be made available to the public.
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

duncan
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Re: Xenodiagnosis in Humans to Detect Bb infection

Post by duncan » Fri 14 Feb 2014 18:49

In this admittedly unscientific mind of mine, this study seems almost counter-intuitive.

First, wouldn't you establish that Xenodiagnosis is a highly effective diagnostic tool in acute Bb infections, i.e. with active EM's? Then progress to acute case after EMs have vanished? Then to disseminated cases? Then to late stage?

And at each step of the Borrelia infection verify the efficacy of Xenodiagnosis in humans with Bb?

Then, and only then, after each step has been validated, then employ it in cases of PTLDS? Why in the world would anyone mix acute cases with late stage and PTLDS BEFORE validating the process through and through?

I am left wondering what the goal of this exercise was.

TicksSuck
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Re: Xenodiagnosis in Humans to Detect Bb infection

Post by TicksSuck » Fri 14 Feb 2014 20:48

Here are some interesting excerpts from the full article:
One subject with PTLDS (D-02) was considered positive in 2 separate xenodiagnostic procedures. Ticks recovered from the initial xenodiagnostic procedure were tested using protocol 1. One nymph was found to be positive by PCR of the nymph lysate culture, but direct PCR of the nymph lysate and microscopic evaluation of the culture were negative.
...
The original positive ospA PCR of the tick culture was confirmed by PCRs for other B. burgdorferi genes. PCR for flaB, ospC, and a second primer set for ospA were positive, but recA PCR was negative. The DNA extracted from this culture sample was then tested by IA/ PCR/ESI-MS, which was positive for 7 of the 8 assay primer pairs. It identified the DNA as from a novel genotype of B. burgdorferi, due to its unique combination of base-count signatures.
...
Xenodiagnosis was repeated approximately 8 months after the original procedure. Direct testing by IA/PCR/ESI-MS revealed that 1 tick was positive for B. burgdorferi, by detection of 2 of 8 assay primer pairs. The 2 base-count signatures detected were consistent with the previously found genotype.
TicksSuck

Martian
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Re: Xenodiagnosis in Humans to Detect Bb infection

Post by Martian » Fri 14 Feb 2014 20:56

duncan wrote:I'm not sure it has been demonstrated it works so well in humans.
I don't know. It may indeed be different in humans. This is a crucial question.

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