1 Three Notable NIAID 2012 Research Projects On Lyme Disease

The National Institute of Allergy and Infectious Disease (NIAID) is conducting some Lyme disease related research which I think readers should know about. There are a number of projects to be found on the Project Reporter web site which may be fascinating, but I took the time to select and highlight a few projects which would be of greater interest to patients suffering with Lyme disease and/or its coinfections.

Project: AN INTRACELLULAR NICHE FOR BORRELIA BURGDORFERI
Institution: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
PI: Skare, Jonathan

Description (by applicant):

Lyme disease, caused by the spirochetal bacterium Borrelia burgdorferi, is the leading arthropodborne infection in the United States and causes significant morbidity in endemic areas. If untreated B. burgdorferi can persistently infect individuals even though the host mounts a potent adaptive immune response such that antibodies obtained from infected patients or experimentally infected animals effectively kills in vitro cultivated B. burgdorferi. In addition, a robust cell-mediated proinflammatory response is observed that induces IL-6, IL-12 and IFN- and inhibits IL-10. Furthermore, the spirochete can resist complement killing demonstrating that this important component of the innate immune response is not sufficient to eliminate B. burgdorferi infection.

The observation that B. burgdorferi persists in such a hostile environment indicates that the spirochete is adept at evading the host immune response via mechanisms that have not been completely elucidated. One possibility is that B. burgdorferi invades host cells and survives at low levels. Recently we have determined that B. burgdorferi invade both immortalized and, more importantly, primary cells (both fibroblasts and endothelial cells) and persist as viable cells in o-culture. In addition we have preliminary data suggesting that the ability to invade host cells involves both integrin binding and Src kinase activity.

In this application we propose to further characterize the internalization of B. burgdorferi and track the fate of B. burgdorferi within thes infected cells to determine how they affect the localized host response following infection. To accomplish this we will use both in vitro correlates of invasion and intracellular survival as well as in vivo imaging of experimentally infected mice as readouts for our studies.

Specifically, we propose to:

(1) Characterize the invasion of Borrelia burgdorferi into primary fibroblasts. The working hypothesis here is that B. burgdorferi exploits invasion as an additional mechanism to avoid host clearance. Our preliminary studies demonstrate that B. burgdorferi invasion is not dependent on host fibronectin, but does involve B1 integrins other than a5B1. In this Aim we will identify the subunit that pairs with B1 to promote invasion and will also evaluate how B. burgdorferi traffics within these cells; and

(2) Determine if invasion is required for B. burgdorferi persistence in vivo. Our working hypothesis is that invasion contributes to persistence by providing an immunoprotected niche for B. burgdorferi. Since Src kinases are required for borrelial internalization in vitro, we will determine whether Src kinase inhibitors alter the infectivity potential of B. burgdorferi in vivo. In addition to standard cultivation and molecuar approaches, novel in vivo imaging will be employed to assess how the inhibitor affects colonization.

The overall goal of these studies is to determine the extent in which an intracellular locale contributes to borrelial persistence.

PUBLIC HEALTH RELEVANCE: Borrelia burgdorferi, the etiologic agent of Lyme disease, is the most common arthropod-borne infectious agent in the United States, and, as such, represents an important Public Health issue. The studies described in this application are designed to address how B. burgdorferi is able to persist effectively in infected mammals despite effective innate immune killing mechanisms and a potent adaptive immune response directed against this pathogen. The hypothesis being tested herein is that B. burgdorferi is capable of low-level intracellular survival in non-immune cells as an additional strategy to prevent borrelial host clearance.

Link: http://projectreporter.nih.gov/project_info_description.cfm?aid=8300386&icde=12284856

Comment: This really begins fulfilling my wishlist, and I look forward to the imaging study videos that I hope will be made and posted online. If there is some sort of confirmation of intracellular Bb in vivo this may explain why some patients need additional antibiotics and why existing treatments may be inadequate as a matter of timing.

This next project is bound to generate discussion, as it involves the potential role of toxins in Borrelia burgdorferi. In this case, the researcher is looking for gene clusters in Borrelia burgdorferi which may create cytolysins similar to the toxins which are found in Staphylococcus aureus, Listeria monocytogenes, and Clostridium botulinum.

Project: A COMMON DENOMINATOR OF PATHOGENESIS; A RARE OPPORTUNITY FOR NOVEL THERAPEUTIC DE(VELOPMENT)
Institution: UNIVERSITY OF ILLINOIS URBANA-CHAMPAIGN
PI: Mitchell, Douglas

Description (by applicant):

Abstract: The 20th century witnessed several major advances in medicine. Perhaps most important were the discovery of antibiotics for bacterial infections and effective vaccines for several major viruses. Unfortunately, the creation of effective vaccines for bacteria has lagged behind analogous anti-viral strategies. Compounded with the rise in antibiotic resistance and a lack of interest from the pharmaceutical industry in pursuing novel antibiotics, we risk losing the fight against bacterial pathogens.

Described herein is an unconventional strategy to exploit bacterial toxins as both novel targets for antibacterial agents and antigens for vaccine development. To intelligently address the increasing threat posed by bacterial pathogens, more effort is needed to uncover the molecular underpinnings of virulence. Our group specializes in the use of bioinformatics, in vitro reconstitution, and genetic manipulation to identify and characterize gene clusters that are responsible for the biosynthesis of virulence-promoting cytolysins. The best-known toxin in this family is the highly modified peptide, streptolysin S (SLS, produced by Streptococcus pyogenes).

SLS production is required for the infective process, but not essential life processes. Our work has uncovered SLS-like toxins are synthesized by at least three other notorious human pathogens, including Staphylococcus aureus, Listeria monocytogenes, and Clostridium botulinum. We aim to study the potential role of the SLS-like toxin in an additional organism, Borrelia burgdorferi (Bb), which causes Lyme disease.

Although widely known, the Bb molecular mechanism of pathogenesis is inadequately defined. If the SLS-like toxin was indeed employed during Bb infections, this would represent the first demonstration of toxin utilization in this family of organisms and would prompt a major revision of borrelioses.

Because bacteria typically employ disparate pathogenic mechanisms, the conserved, SLS-like pathway provides a rare opportunity to develop more broadly applicable, yet targeted countermeasures. From our perspective, new antimicrobial strategies should directly target the pathogenic mechanism, rather than DNA replication, protein synthesis, or the cell wall. This approach holds enormous potential, as these drugs will theoretically be resistant to resistance.

This project will identify inhibitors of SLS toxin biosynthesis for the specific purpose of developing novel antibacterials. Moreover, SLS is non-immunogenic, rendering it an unfeasible candidate for vaccine development.

We have succeeded in generating attenuated variants with the anticipation that these can be used for raising toxin-neutralizing antibodies. The notion of immunizing against a bacterial toxin represents a potentially general strategy for future vaccine development.

With this proposal, we aim to not only fundamentally shift the accepted view of Bb pathogenesis, but also to challenge the paradigm that antibiotics must kill bacteria and non-immunogenic toxins are intractable vaccine candidates. These seemingly unrelated goals are actually quite intertwined. Our approach rests on the philosophy that a more complete understanding of toxin biosynthetic pathways and chemical structure can be rationally exploited to design novel therapeutics.

Public Health Relevance: Bacterial pathogens employ numerous mechanisms to evade the human immune system. We have discovered a novel strategy within the organism that causes Lyme Disease, who's pathogenesis remains largely enigmatic. A greater understanding of these processes will lay the foundation for developing the next generation of antimicrobial drugs.

Link: http://projectreporter.nih.gov/project_info_description.cfm?aid=8145943&icde=12284856

Comment:

Wait... I thought Radolf & co. said Borrelia burgdorferi does not produce a toxin? I know Donta patented some genes in Bb he saw as being analogous to a toxin.

Is there now evidence of newly researched genes which create a toxin in Bb? Or is this an old hypothesis which is being revisited?

Project: ASSESSMENT OF PATIENTS WITH BORRELIA INFECTION
Institution: NIAID
PI: Marques, Adriana

Description (by applicant):

Lyme disease is a multisystem illness caused by infection with the spirochete Borrelia burgdorferi and it is the leading vector-borne disease in the United States. Our current work addresses the following areas in Lyme disease: development of new tests and biomarkers for infection, investigation of persistence of infection with B. burgdorferi in humans, search for the cause of Southern Tick-associated Rash Illness (STARI), and investigation of the role of immune response in Lyme disease and PLDS.

One of the main problems in Lyme diagnosis has been the lack of highly specific and sensitive assays for B. burgdorferi and the lack of a test that could be used to assess response to therapy. Such assays should greatly facilitate the accurate diagnosis of Lyme disease and assessment of response to therapy in individual patients. Currently, no such test is available.

We have developed a new test using the luciferase immunoprecipitation systems (LIPSs) for profiling of the antibody responses to a panel of B. burgdorferi proteins for the diagnosis of Lyme disease. A synthetic protein consisting of a repeated antigenic peptide sequence, named VOVO, had the best diagnostic performance, similar to the C6 test (a diagnostic test using a peptide ELISA that we have helped develop and is highly sensitive and specific). The VOVO LIPS test displays a wide dynamic range of antibody detection spanning over 10,000-fold without the need for serum dilution; and offers an efficient quantitative approach for evaluation of the antibody responses in patients with Lyme disease.

Recent studies have shown that B. burgdorferi may persist in animals after antibiotic therapy and can be detected by using the natural tick vector (Ixodes scapularis) to acquire the organism through feeding. Whether this occurs in humans is unknown.

We have implemented a new clinical protocol to investigate the utility of this approach for identifying persistence of B. burgdorferi in treated human Lyme disease.

STARI is a rash similar to the rash of Lyme disease that occurs in persons residing in southeastern and south-central states and is associated with the bite of the lone star tick, Amblyomma americanum. The cause of the rash is unknown, as it is the natural course of the disease.

We have a clinical protocol to investigate the cause of STARI, and we are applying new genomic tools that identify bacteria based on species-specific sequences in the 16S rRNA ribosomal genes to the skin biopsies from patients with STARI.

Inflammatory innate immune responses are critical in the control of early disseminated infection, while adaptive immune responses are vitally important, particularly the humoral immune response, in controlling spirochete levels in tissues and resolution of Lyme arthritis in animal models. We are examining the antibody response to immunogenically dominant antigens of B. burgdorferi in PLDS patients and controls.

Further investigation of the anti-borrelia immune response may help in elucidating the pathogenic mechanism of PLDS and yield important information for future approaches to diagnosis and treatment. We have a clinical protocol in which we use DNA microarrays to characterize gene expression patterns in skin biopsies from individuals with EM, with the aim of capturing the human host response to pathogen exposure.

We are also investigating the differences in immunological response between predominantly lymphocytic meningitis and predominantly neutrophilic meningitis. Results from these studies will serve as a window into the fundamental biology of the infection.

Link: http://projectreporter.nih.gov/project_info_description.cfm?aid=8336099&icde=12284856

Comment:

The existence of the VOVO LIPS test is nothing new - reports on the development of this test have been around since 2010. Also, there is already information about a chronic Lyme disease xenodiagnosis study out there.

It seems like this project has a large scope - or consists of more than one project under the same umbrella. So far, no project end date has been posted for this entry.

What would be of most interest to me would be finding differences in immunological response between patients with acute Lyme disease and those with assumed PLDS - something Alaedini has already been studying.

(Side note: I thought that it was already determined that Borrelia lonestari, a relapsing fever spirochete, was the cause of STARI or Masters disease - did I miss something?)

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10 Dr. Barthold Discusses Persistence On Diane Rehm Show

Several days ago,  Dr. Barthold was interviewed on the Diane Rehm Show on NPR. A full transcript to that show can be found on the WAMU radio/NPR web site, if you are interested in seeing what Dr. Auwaerter, Dr. Fallon, and Dr. Shor had to say about chronic Lyme disease.

I find what Dr. Barthold had to say about persistence to be very interesting.

(EDIT: Unfortunately, the strict terms about republishing any portion of the transcript were pointed out to me and I had to remove Barthold's quotes from this page - please refer to the transcript above in following what I say below.)

Did anyone else listen to the show or read the transcript and catch what he said about this:

The remaining organisms - potentially these persister cells - are in connective tissue and not eliciting inflammatory change. He sees very little inflammation in the animals he has tested and in which he found persistent spirochetes.

Yet when he removes the bacteria from a mouse and puts it in a new mouse, the spirochetes cause inflammation all over again.

What's up with that? Pretty strange, isn't it?

Can one form a hypothesis about why this is happening or at least take a shot at it?

I have a few ideas about this and will be putting them in comments in this post over the next few days, providing readers with the disclaimer that they are hypothetical and not to be taken as confirmed fact. Someone else will need to do the research on this issue.

Does anyone else here reading along have their own hypothesis about why this is happening - why after antibiotic treatment, he found persistent bacteria in these animals - yet they are not causing inflammation?

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11 Why Aren't Persisting Spirochetes Enough Evidence Of Infection?

On the heels of Embers et al having published their statement on PLoSONE, a number of patients are already questioning its content.

Some are claiming that Embers et al statement about how their findings should not be used to oppose current IDSA treatment guidelines for Lyme disease is something they were asked to write - rather than something the authors included on their own.

I don't know. For this claim - whether it's true or not - I have no evidence. However, one thing I do know is that there are solid scientific reasons which back the need for more research on spirochetes which survive after prolonged antibiotic treatment.

The question, of course, which weighs heavily on every patient's mind has been this one:

Why aren't persisting spirochetes enough evidence of infection?

It's become a political hot button question, and it's a scientific question. But most people think that as long as the spirochetes Embers found are alive and metabolically active, that is enough evidence to state that yes, Lyme disease is a chronic infection - let's stop all this nonsense right now and change the treatment guidelines!

Given my own experience and how longer than standard treatment helped me improve, I totally get this. I've been there, done that - and I think that a standard course of antibiotic treatment does not work for everyone. Particularly if there is a delay in proper diagnosis and treatment. Particularly if a coinfection is present. Particularly if there is some abnormality in one's immune system.

But if you are a scientist and you are researching this phenomenon of persistence - whether you as a scientist suspect these spirochetes can cause persisting infection or not; whether the above claim by other patients is true or not - you will be called upon by other scientists to support your findings.

It isn't just going to be the IDSA or the ALDF or other organizations which deny the possibility of persistent infection as a cause of chronic Lyme disease which are going to want to know the outcome of your study.

It's going to be the American Society for Microbiology (ASM) that wants to know the outcome. It's going to be researchers in Europe like the Brorsons who study the "cyst" form of Borrelia burgdorferi and want confirmation of their own findings about persistence.

It's also going to be universities and health departments and many different organizations which may not have any particular position on whether or not Lyme disease can be chronic who will want to know the outcome of your study.

They're all going to want to know the outcome of a study such as Embers et al, so these researchers must be certain about what they found and its significance, and conduct additional research related to their findings in order to confirm them.

They must find evidence that no one can argue against - even the most skeptical - if they are to support their own hypotheses. And it may be that at this stage they genuinely do not know what to make of these persistent spirochetes and not only their ability to cause disease - but how they cause it.

I can easily imagine that Embers et al is being very cautious about the interpretation of their results and wanting further studies as easily as it is for other people to imagine that Embers at al were somehow instructed to downplay the significance of their spirochetes surviving antibiotic treatment.

Why do I say this? I say this because I have learned a few things about these stealthy bacteria and think there is good reason for Embers et al to be cautious about the interpretation and approaching their results either way.

Borrelia burgdorferi spirochetes, plasmids, and infectivity

After doing some research on this issue, the issue of whether or not these spirochetes were infectious and pathogenic or not is a more complex issue than it at first appears.

First, here's a refresher of some basic microbiological definitions. (Bear with me, I'll try to get through this part quickly.)

Infection = the replication of organisms in the tissue of a host; when defined in terms of infection, disease is overt clinical manifestation. In an inapparent or subclinical infection, an immune response can occur without overt clinical disease.

Colonization = A carrier (colonized individual) is a person in whom organisms are present and may be multiplying, but who shows no clinical response to their presence.

Pathogenicity = The pathogenicity of an agent is its ability to cause disease; pathogenicity is further characterized by describing the organism's virulence and invasiveness.

Virulence = refers to the severity of infection, which can be expressed by describing the morbidity (incidence of disease) and mortality (death rate) of the infection.

Invasiveness = invasiveness of an organism refers to its ability to invade tissue.

Now that we're past these definitions, I'll cut to the chase and say there are two important things to know upfront:

1) During in vitro passage or certain stressors, Borrelia burgdorferi can lose some of their plasmids. How soon this happens varies depending on the strain and particular isolates of Borrelia. 

2) When Borrelia burgdorferi loses specific plasmids with specific genes on them, it can lose infectivity and pathogenicity. It should be noted that specific genes for specific purposes can show up on different plasmids on different strains. (For example: Bb strain N40's VlsE locus is different from the one found on commonly studied B31, and it shows up on a different plasmid than on B31.) 

The essential bit of information here is that the loss of a particular gene or set of genes can affect spirochetes' ability to cause infection - and even if these genes are lost, spirochetes may still survive for a while. They can become attentuated or less infectious.

Numerous studies on Borrelia burgdorferi's plasmids have shown that lp28-1 is a linear plasmid which makes Borrelia burgdorferi infectious. VlsE genes found on lp28-1 are thought to be essential for mammalian infection with Borrelia burgdorferi.

When the lp28-1 and yet a different plasmid, lp25, are missing from spirochetes, they are unable to infect mice. The lack of lp25 completely abolishes infectivity since this plasmid encodes a gene (bbe22) which is essential for Borrelia burgdoferi's survival in mice.

Spirochetes which lose lp28-1 plasmids will still live for a while - but the immune system tends to mop them up in a few weeks without antibiotic usage.

Specific research on mutant spirochetes with a lack of the lp28-1 plasmid has shown the following:

"While the wild-type B. burgdorferi persisted in tissues for the duration of the study, the lp28-1− mutant began clearing at day 8, with no detectable bacteria present by day 18. As expected, the wild-type strain persisted in C3H/HeN mice despite a strong humoral response; however, the lp28-1− mutant was cleared coincidently with the development of a modest immunoglobulin M response. The lp28-1− mutant was able to disseminate and persist in C3H-scid mice at a level indistinguishable from that of wild-type cells, confirming that acquired immunity was required for clearance in C3H/HeN mice. Thus, within an immunocompetent host, lp28-1-encoded proteins are not required for dissemination but are essential for persistence associated with Lyme borreliosis."

To translate the above:

Normal Bb spirochetes infected C3H/HeN (mice which are specifically bred for the ability to demonstrate joint swelling and arthritic symptoms similar to those found in the average person who gets Lyme disease) mice and these spirochetes could not be cleared by the immune system despite the fact that these mice had a strong humoral response.

However, mutant Bb spirochetes which did not contain linear plasmid 28-1 were completely cleared by these C3H/HeN mice.

What's fascinating about this study is even though the mutant Bb spirochetes lacked lp28-1, these spirochetes could still disseminate. Only in severely compromised immune deficient mice (scid mice) could the spirochetes both disseminate and persist - acquired immunity must be functional in animals infected with such mutants in order to clear the spirochetes.

So, here is one example of how you can have spirochetes which are alive and metabolically active and  can even disseminate - yet they are no longer causing disease. In this instance, they were cleared by the immunocompetent mice without the use of any antibiotics within a mere 18 days. (I wish I were that lucky!)

More recently, other plasmids have been found to contribute to infectivity in mammalian hosts - such as lp36. lp36 is viewed as being another major contributor to persistent infection in mice, and spirochetes become attenuated when lp36 is removed.

Linear plasmid 28-1 and lp25 have a much longer history of their role in infectivity and pathogenicity, and they are two of the most studied linear plasmids thus far - lp28-1 the most because of its VlsE genes in strain B31.

So, keep this in mind when you think of the Embers et al study, and realize why this part of their paper on Rhesus macaques caught my attention:

"A few spirochetes grew in cultures of organ tissues collected post-mortem from each animal after  > 9 weeks, but we were unable to subculture any spirochetes from either treated or untreated animals due to their slow growth. We therefore pelleted these cultures to confirm their identity and test their viability by DNA/RNA analysis. Transcription was detected in culture pellets and the tissues of treated animals, indicating that the bacteria were metabolically active (Figure 6C, D). Figure 6D shows ospA transcription detected directly in tissues harvested from treated and untreated animals. We also hypothesized that persistent spirochetes may lose linear plasmid 28-1 (lp28-1), which encodes the VlsE antigen bound by the anti-C6 antibody. Transcription of a lp28-1 gene (bbf26) was verified in organ tissue from both untreated animals and one treated animal (Figure 6D).

In the case of Embers et al study on Rhesus macaques, one antibiotic treated animal was found to have evidence of transcription of a lp28-1 gene (bbf26 - protein; purpose unknown) from a sample taken from heart tissue (Fig. 6D) and that transcription should only be able to occur if the lp28-1 plasmid is intact and functional. lp28-1 is a linear plasmid which is very specific to infection both in vitro and in vivo, whether a tick or needle inoculation is used.

In Embers study, in addition to transcription of a gene from lp28-1, OspA transcription from lp54 was found in three treated animals. OspA transcription was detected in two tissue samples taken from the bladder and one tissue sample taken from the spleen. Additional OspA transcription was found in different organs in two out of three of the same animals using organ tissue culture pellets.

Overall, this sounds interesting and points to the possibility of chronic infection after antibiotic treatment.

 But if I have seemed cautiously optimistic about this study, it's because of a few factors*:

1) Only one treated animal had evidence of a infection where lp28-1 transcription was taking place - had more treated animals shown evidence of transcription on this plasmid, I would have been more excited. How long could spirochetes maintain these plasmids while being treated? What about lp25?

2) It is unknown to me if the genetic background and/or immune system of the treated Rhesus macaques somehow played a role in their inability to clear the spirochetes which remain after antibiotic treatment. (Refer to this post on HLA-DR types, read what's before and after the "=" signs, and you'll see what I mean.)

3) it is unknown to me how different the results would be if the Rhesus macaques had been infected using ticks instead of needle inoculations. It seems to make sense to me to do this study again using ticks because that mimics what happens in nature.

On the other hand, I find it very interesting that three animals showed evidence of transcription of OspA. Given how much inflammation people experience during Lyme disease - plus evidence of later stage antibody reactivity to OspA - it at least gives me pause to think about how often OspA has been a culprit for my own symptoms, directly or indirectly.

The only kind of spirochete
you don't mind getting close to.

So it's a mixed bag how I look at the results of the Embers Rhesus macaque study. I think it's a positive step in the right direction establishing what happens with spirochetes in their host after antibiotic treatment. And yet the unanswered questions for me seem related to the same unanswered questions the researchers themselves wrote in their paper.


Is There Anything Positive To Glean From Dr. Baker?


Of Dr. Baker's two major stated issues with the Embers study, the only one now left is whether or not the spirochetes which were transmitted by ticks to new hosts (xenodiagnosis) were in fact infectious. His other concern was over the use of ceftiofur in the study rather than ceftriaxone - however, the authors of the study have since posted a correction to PLoSONE stating that ceftriaxone - not ceftiofur - was used throughout the entire study.

If there are any remaining minor issues he has with the study, he has yet to share them on the Lyme Policy Wonk blog. Mostly, he seemed to reiterate his concern about these two issues and focused on the single mention of ceftiofur in the paper repeatedly.

About the most positive response I heard from Dr. Baker on that blog thus far was about his view of how Lyme disease research should be conducted:

"...I favor a multi-disciplinary approach that moves the field in a different direction, rather than solutions based on the assumed yet to be proved existence of a persistent infection that can only be cured by antibiotics. I don’t really discount such a view; rather, I feel we are neglecting other possibilities that may provide the answers we all are looking for. A case in point, would be the recent work of good friend, Armin Alaedini — who I helped support when I was at the NIH– using specimens collected by Mark Klempner as part of his clinical trial. These valuable specimens are being maintained by Mark in a specimen repository for use in just such cutting-edge research. They are available free of charge on request."

Like Pamela Weintraub, I agree that a multi-disciplinary approach to research on Lyme disease is important. And while Dr. Baker also supports a multi-disciplinary approach to research on Lyme disease and he states he doesn't discount the view of persistent infection in the above paragraph - his direct responses to patients suffering with CLD/PTLDS state that most patients are suffering from some other non-Lyme disease related condition - something I find particularly unhelpful to my situation. That and a lack of sufficient research on other treatment approaches has been an issue for ages.

In my opinion, Dr. Baker's response to the Embers Rhesus macaque study was more negative than it warranted. I wouldn't have viewed it negatively at all - I see it as a stepping stone in getting a better understanding about Lyme disease.  And just because it leaves unanswered questions does not mean it was inherently flawed - which was what Dr. Baker seemed to suggest.

To quote someone else on that blog:

"My question to Dr. Baker is why don’t you and your colleagues offer some expert advice, according to your best opinions and hunches if science really has proven inadequate for your epistemic standards of validity, without having to officially disclose any sensitive data that might get you in trouble with your career, that could actually HELP these affected people lessen their pain and disability? Just disparaging some controversial or technically flawed research as being invalid does not seem helpful enough to me."

Yes. This.

Regardless of anyone's opinion - Dr. Baker, or LLMDs, or my friends and family - researchers will be expected to provide evidence to the world that these remaining spirochetes are pathogenic. They will need to provide evidence that that they can cause infection and reproduce - even if they are already proven to be alive.

Researchers who are trying to work without bias will want to cover all the bases and check their postulates twice to be 100% certain that Borrelia burgdorferi either causes a chronic infection or it does not after standard antibiotic treatment.

This may be so - but I'm impatient about it.


References:

The Absence of Linear Plasmid 25 or 28-1 of Borrelia burgdorferi Dramatically Alters the Kinetics of Experimental Infection via Distinct Mechanisms. Maria Labandeira-Rey, J. Seshu, and Jonathan T. Skare. Infect Immun. 2003 August; 71(8): 4608–4613. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC166013/

Correlation between plasmid content and infectivity in Borrelia burgdorferi. Purser JE, Norris SJ. Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13865-70. http://www.ncbi.nlm.nih.gov/pubmed/11106398

High- and low-infectivity phenotypes of clonal populations of in vitro-cultured Borrelia burgdorferi. Norris, SJ, Howell, JK, Garza, SA, Ferdows, MS, and Barbour, AG. Infect. Immun. 63:2206-2212.

Plasmid Stability during In Vitro Propagation of Borrelia burgdorferi Assessed at a Clonal Level. Dorothee Grimm, Abdallah F. Elias, Kit Tilly and Patricia A. Rosa. Infect. Immun. June 2003 vol. 71 no. 6 3138-3145 http://iai.asm.org/content/71/6/3138.full

Experimental assessment of the roles of linear plasmids lp25 and lp28-1 of Borrelia burgdorferi throughout the infectious cycle. Grimm D, Eggers CH, Caimano MJ, Tilly K, Stewart PE, Elias AF, Radolf JD, Rosa PA. Infect Immun. 2004 Oct;72(10):5938-46. http://www.ncbi.nlm.nih.gov/pubmed/15385497

The critical role of the linear plasmid lp36 in the infectious cycle of Borrelia burgdorferi. Mollie W Jewett, Kevin Lawrence, Aaron C Bestor, Kit Tilly, Dorothee Grimm, Pamela Shaw, Mark VanRaden, Frank Gherardini, and Patricia A Rosa. Mol Microbiol. 2007 June 1; 64(5): 1358–1374. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1974800/?tool=pubmed

Basic Epidemiology. Beaglehole R, Bonita R, Kjellstrom T. World Health Organization, Geneva, Switzerland, 1993

* Factors which concern others but I did not originally think of are included in comments below.

[Edited March 9, 2012 - Removed item above about brain tissue after reviewing Embers paper again - multiple brain samples were taken; one treated animal was positive for B. burgdorferi RNA in both heart and brain.]

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64 Embers et al Issues Statement On PLoSONE

Embers et al has issued the following author statement on PLoSONE:

Persistence of Borrelia burgdorferi in Rhesus Macaques Following Antibiotic Treatment of Disseminated Infection

Author statement
Posted by membersla on 02 Mar 2012 at 15:19 GMT

We recently published this article entitled “Persistence of Borrelia burgdorferi in Rhesus Macaques Following Antibiotic Treatment of Disseminated Infection.” The subject and content of this work may be viewed very divergently, given the controversy surrounding Lyme disease treatment. Specifically, the phenomenon of post-treatment Lyme disease syndrome and its cause (s) have been viewed with much contention.

Our work, which was funded by several grants from the National Institutes of Health, is composed of two major experiments. Experiment 1 entailed a very comprehensive and time-consuming study that indicated the possibility that spirochetes could persist after antibiotic treatment, but only nucleic acid or antigen of these bacteria were detected, leaving open the question of persistence by intact organisms. Experiment 2 was intended to answer that question. The authors elected to publish these 2 studies together, as they mutually enhance the validity and scientific merit of the work.

In our study, we provide evidence demonstrating the post-treatment persistence of the B. burgdorferi spirochete that has been reported previously in a mouse model. We further demonstrate through multiple detection methods that intact spirochetes can survive antibiotic treatment in a nonhuman primate host. It is not our intent to present data in opposition of current antibiotic treatment regimens for humans, but rather to report what we believe to be objective, well-performed experiments on antibiotic efficacy in a nonhuman primate model.

These data are by no means a referendum on long-term antibiotic therapy, nor should they serve to oppose current IDSA guidelines for the treatment of Lyme disease. From the medical standpoint, these results may or may not warrant testing of additional treatments or regimens. This depends heavily on the results of further inquiry as to the duration of persistence, the viability and phenotype of persistent organisms, and the answer to the key question of whether persisters are pathogenic. Current practices could only be challenged by solid proof of better treatment options; these are currently not available.

For several decades, basic scientists and medical doctors have collaborated to understand and improve Lyme disease treatment, diagnosis and prevention. As we proceed with further inquiry into the phenomenon of PTLDS, these collaborations are essential. The continued discussion, commentary and debate will additionally be of benefit when conducted without bias.

Source Link: http://www.plosone.org/annotation/listThread.action?inReplyTo=info%3Adoi%2F10.1371%2Fannotation%2Fbe820481-edcb-457e-8d20-c0a904b91607&root=info%3Adoi%2F10.1371%2Fannotation%2Fbe820481-edcb-457e-8d20-c0a904b91607

Bolded emphasis mine.

My comments? I could have predicted that such a statement would be issued. The researchers in question are making a string of inquiries where they must be certain of the outcome and not make a statement about the nature of Lyme disease prematurely - even if the chronic infection model of Lyme disease makes sense to many people and is the experience which a number of patients report having.

It comes as a disappointment to many patients that there is a specific statement here against long term antibiotic treatment, with the explicit statement that the results of this study are not intended to oppose current IDSA treatment guidelines. However, it is also stated that "these results may or may not warrant testing of additional treatments or regimens". The future regarding changes in antibiotic usage is uncertain.

Statements above and within the original study reflect a certain amount of uncertainty.

And in order for this team to avoid becoming cargo cult scientists, they must be willing to look at all the possible causes for persisting symptoms. They must be willing to challenge themselves and question the strength of their suspicions and poke holes at their assumptions. This must be done not only to assure them they are on the right track - but to be prepared to answer questions coming from any critics.

They may repeat their experiments to confirm their findings and conduct another study where tick inoculation is used and not needle inoculation. A study is needed that examines what would happen in an actual case of infection under conditions found in nature - not in a lab. This experiment would not only involve the use of ceftriaxone and doxcycline - but use infected ticks on hosts and involve the complex immune interactions that take place after a tick infection which do not occur the same way as they would after injection with a bacteria-laiden needle.

I'm looking forward to more research from Embers et al. That they close their statement with the phrase, "The continued discussion, commentary and debate will additionally be of benefit when conducted without bias," means a lot to me. It indicates to me that they are seeking the truth - which is something I can get behind. I only hope they find it soon.

Image credit: Morphology of Borrelia burgdorferi by Jeffrey Nelson, Microbe Library. Use under Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

This work by Camp Other is licensed under a Creative Commons
Attribution-NonCommercial-ShareAlike 3.0 Unported License.

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0 Books: Tickborne Diseases and Disease Vectors

I've just been informed I'm getting two books in the mail in about a week or so, and when they get here, I'll be sure to let everyone know and offer my own review of them once I've read a significant portion of them...

Tick-Borne Diseases of Humans edited by Jesse L. Goodman, David T. Dennis, and Daniel E. Sonenshine.

Amazon product description: A ready resource, this book covers key information that will be highly useful to students and professionals in the fields of human and veterinary medicine, public health, medical entomology, acarology, and ecology. Written by experts with specialized field knowledge, "Tick-Borne Diseases of Humans" presents state-of-the-art information on disease epidemiology, transmission, and ecology.

The book is divided into three sections, each of which can be used independently or in concert with the remaining two sections.

 Section I integrates divergent information relevant to the full spectrum of tick-borne diseases, incorporating tick biology and identification, distribution of the diseases ticks transmit, and various strategies for tick control. In addition, this section comprehensively reviews the clinical approach to a patient with a possible tick-borne affliction.

Section II is devoted to in-depth profiles of specific diseases, including information on disease history, biology, epidemiology, ecology, transmission, clinical manifestations, diagnosis, treatment and prevention. And, Section III examines the geographical distribution of tick-borne diseases and their vectors.

This book examines the striking increase in incidence and our subsequent awareness of a broad array of tick-borne diseases.

It addresses both vector and disease perspectives, including state-of-the-art information on disease epidemiology, transmission, and ecology; clinical and laboratory findings; diagnosis; and treatment and prevention.

It includes a useful full-color insert, with maps of vector and disease distribution, an atlas of clinical and pathologic images, and illustrations of diagnostically important skin lesions and blood smears; introduces public health practitioners, research scientists, and students to the field and also provides references for information beyond traditional areas of expertise; and also presents accessible information to an informed public on disease transmission, clinical laboratory diagnosis and treatment, and history of infections.      

The Biology of Disease Vectors by Barry J. Beatty and William C. Marquardt. (This is the first edition, and I'll be getting the second edition shortly thereafter to make a comparison.)

This comment is stated on the second edition on Elsevier: "Biology of Disease Vectors presents a comprehensive and advanced discussion of disease vectors and what the future may hold for their control. This edition examines the control of disease vectors through topics such as general biological requirements of vectors, epidemiology, physiology and molecular biology, genetics, principles of control and insecticide resistance. Methods of maintaining vectors in the laboratory are also described in detail. No other single volume includes both basic information on vectors, as well as chapters on cutting-edge topics, authored by the leading experts in the field. The first edition of Biology of Disease Vectors was a landmark text, and this edition promises to have even more impact as a reference for current thought and techniques in vector biology."

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1 Persistence of Borrelia Infection After Antibiotic Use In Mice

For the random passerby who types "is chronic lyme real" into Google, here is more data to consider, coming from Columbia University.

Credit to: Dr. Brian Fallon, Lyme and Tick-borne Diseases Research Center, Columbia University.
Link source as of Feb. 17, 2011: http://www.columbia-lyme.org/research/keyarticles.html

Persistence of Borrelia burgdorferi in mice after antibiotic therapy.

Two studies have recently been published which reveal that Bb may persist in the mouse despite antibiotic therapy. These studies support much earlier work by Straubinger et al in the dog model (1997) and Bockenstedt et al (2002) in the mouse model. Bockenstedt et al (2002) showed that Bb persistence can occur after antibiotic treatment and that these spirochetes could be acquired by ticks (xenodiagnosis) but that the infected ticks could not transmit infection to naïve hosts – suggesting that the spirochetes were attenuated in that they had become non-infectious. Straubinger et al (1997) had shown that even after 30 days of antibiotic treatment, Bb spirochetes could be demonstrated in 3/12 dogs by culture, and DNA could be demonstrated by PCR in 9/12 dogs long after treatment.

More recently, Hodzic et al from UC Davis in California reported in Antimicrob Agents Chemother. (2008;52(5):1728-36) a study which examined the effectiveness of antibiotic treatment using ceftriaxone or saline for 1 month. Mice were treated either early in the infection (3 weeks) or later (4 months). Tissues were tested by immunohistochemistry, PCR, culture, transplantation of allografts, and xenodiagnosis at 1 and 3 months after treatment. Tissues from the mice treated with antibiotics were culture negative, but tissues from some of the mice remained PCR positive and intact antigen-positive organisms with spirochetal morphology were visualized in collagen-rich tissues. Xenodiagnosis demonstrated that uninfected larval ticks after feeding on the antibiotic-treated mice were able to acquire spirochetes (confirmed by PCR) and then transmit these spirochetes to naïve SCID mice which became PCR positive but culture negative. This study therefore demonstrated that antibiotic treatment in the mouse model does not result in eradication of the Bb spirochetes and that some of these spirochetes were infectious, although attenuated in activity.

Yrjanainen et al from Univ of Turku in Finland reported in J Infectious Disease (2007; 195(10):1489-96) a study which examined whether anti-tumor necrosis factor-alpha would have a beneficial effect on Bb-infected mice. C3H/He mice were infected with B. garinii A218 or B. burgdorferi sensu stricto N40. In study 1 (with B. garinii) and in study 2 (with Bb SSN40), 2 weeks after infection, 10 mice were treated with ceftriaxone only for 5 days and 10 mice were treated with anti-TNF-alpha only. In another group of 10 mice, anti-TNF was added simultaneous to the ceftriaxone at 2 weeks after infection while in another group of 10 mice anti-TNF was added at 6 weeks after infection (ie, 4 weeks after ceftriaxone). Finally, a fifth group of mice was treated with saline as a sham treatment. For the group that received ceftriaxone only, no samples were positive by culture or by PCR at 2 weeks after infection. However, among those mice treated with anti-TNF-alpha either at 2 weeks or 6 weeks after infection, spirochetes grew from one-third of the mice. Contrary to earlier findings by Bockenstedt et al (2002) in which the spirochetes detected after antibiotic treatment were attenuated in activity, the recovered spirochetes in this study did not appear to be attenuated, as ceftriaxone sensitivity rates, plasmid profiles, and virulence rates were similar to those of bacteria used to infect the mice. This study demonstrated that a portion of B. burgdorferi-infected mice still have live spirochetes in their body, which are activated by anti-TNF-alpha treatment.

Commentary

 These two studies demonstrate that Bb spirochetes can persist in the mouse after ceftriaxone therapy. The Finnish study was remarkable in that culture and PCR were negative after ceftriaxone but, after additional treatment with anti-TNF-alpha, viable spirochetes were recovered. TNF is a pro-inflammatory cytokine which, when blocked, typically results in a reduction in clinical inflammation; for this reason, such treatment is used for patients with rheumatoid arthritis. To the surprise of the authors, viable spirochetes were recovered in these PCR- and culture-negative mice after TNF blocking treatment was given. Also interesting is that anti-TNF treatment did not result in the expected finding of a reduction of joint swelling.

The Finnish study was the first study to demonstrate that immunomodulatory treatment of animals infected with Bb could convert them from culture negative to culture positive. The California study was remarkable in that only tick-feeding was capable of extracting infectious but non-replicating attenuated spirochetes; without having done that step of xenodiagnosis and then transferring the tick to feed on naïve SCID mice, the authors’ conclusion would have been that infectious spirochetes do not persist in the mouse model as culture was negative. The authors further concluded that negative culture and PCR can not be relied upon as markers of treatment success.

We do not know the extent to which these findings can be translated to the human situation. Nevertheless, the activation of infectious spirochetes after anti-TNF therapy in mice should alert clinicians to the possibility that anti-cytokine therapy may result in a similarly increased risk of activating latent infection among patients with a history of treated Lyme disease. At this point, we do not know whether attenuated spirochetes are capable of inducing illness-symptoms in mice or humans; while it is possible that spirochetal mRNA may be producing surface lipoproteins that stimulate systemic symptoms, this hypothesis needs to be tested in the next phase of this important research.

BAFallon, MD

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So here is food for thought. I would like to see these studies repeated by someone else for confirmation of these findings. Of course, this is in mice and not in humans, but I've said before, mice have been used by researchers for ages now to determine disease courses and treatment possibilities in humans. 


One thought after this is that if there is an immune dysregulation element to persistent infection, it may be harder to treat that aspect of it if treatment itself activates a latent infection. 


One next step for discovering how Borrelia burgdorferi persists would be to carry out  xenodiagnosis in human hosts based on the first study above - of which a similar study, Searching for Persistence of Infection in Lyme Disease, is being conducted by the NIH. This study has been received with some element of controversy, though - and will be discussed in a future post.

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