:  “… the most prominent difference was the virtual absence of reactivity with OspA 

and OspB in German patients.  It is possible that the sera of these patients contained 

antibodies to other epitopes of OspA or OspB than those present in the recombinant proteins, 

but their sera almost always lacked reactivity in the 31- to 35- kDa region with antigens from 

sonicated whole spirochetes of all three strains.  In contrast, in a previous study of 127 US 

patients with various manifestations of Lyme disease, 71% of the 80 patients with arthritis 

had strong IgG reactivity with OspA or OspB or both that developed near the beginning of 

prolonged episodes of joint involvement, from 5 months to 7 months after disease onset [25].  

The combination of the HLA-DR4 specificity and OspA and OspB reactivity was associated 

with chronic arthritis and lack of response to antibiotic therapy.”  Dressler F, Ackermann R, Steere AC. ,  

Antibody responses to the three genomic groups of Borrelia burgdorferi in European Lyme borreliosis. J Infect Dis. 

1994 Feb;169(2):313-8.  PMID: 8106763 [PubMed - indexed for MEDLINE]

As you know, OspA and B are left out of the US criteria.  In his first analysis, he said OspB and 41 were the consistent 

early antibodies (1986).  They conducted this trial on children in Europe, anyway, knowing it was useless:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10547245

Dr. Radolf says he's skeptical of Dr. Harvey's theories linking the Lyme disease bacterium with other ailments. Dr. Radolf has done extensive research in Lyme disease and diseases caused by Borrelia burgdorferi and other bacteria.  

"Lyme disease does have neurological syndromes," he says. "But regarding neurological diseases such as ALS and MS, I think very few people in the neurological community would accept that these are due to Lyme disease," or Borellia [sic] burgdorferi.  I don't believe there is any evidence that real, properly diagnosed ALS is caused by Borellia or that it is treatable with antibiotics."     -- Dallas Morning News, April 30, 2005

---------------------

RECOMMENDATIONS for RHODE ISLAND’S TBDs COMMISSION, APRIL 2002   

A MANAGEMENT PLAN

 TBDs MANAGEMENT OBJECTIVES

1.       A  statewide physician education program is clearly necessary to patient management.

2.      Improved surveillance for known and new TBDs and the development of a sentinel TBDs identification database and DNA ident/sequencing

3.      Immediate testing improvement measures via the discontinuation of the use of  laboratores by RI hospitals and independent physicians, which fail to report correctly, or use even the current US recommended stains.

4.      The development of a patient/pathology database to identify cohorts of patients who would be eligible for neuroprotective regimens such as MMP inhibition, therapeutic kynurenines, or whatever is on the horizon in new antibiotic trials, and blood brain barrier damage, physical, and cognitive rehabilitation after resolution of infections, when or if that becomes possible.

 INTRODUCTION

 Treatment Failed Before.

 That treatment often “fails” in borreliosis is really an incorrect way to frame the problem of Chronic Lyme disease.  Allen Steere discovered and published that persisting spirochetes are responsible for continued Lyme arthritis in 1985 and 1994 (1, 2).  Low levels of spirochetal infection driving an exaggerated immune response that is not completely suppressed with antibiotics, is the subject yet to be understood well enough to be managed adequately.

Some people have more illness symptoms than others, after exposure to Borrelia burgdorferi (Bb). Chronic neurologic Lyme exists, or there would not be an outcry for help with difficulties with diagnosis and being treated for this chronic illness.  The overwhelming evidence from of studies of all types of spirochetal infections in all kinds of animals (except lizards) is that in some mammals, the infection persists despite antibiotic treatment, not primarily due to acquired antibiotic resistance.  In swine medicine, persistently infected animals are called carriers.  Carriers are culled from the herd.  Chronic Lyme patients tend to receive less severe special treatment, but they are deliberately made to become medical and social outcasts.  The state of Rhode Island now seems to recognize the magnitude of the problem, and the patients are appreciative.

In the early years since the discovery of the Dr. Burgdorfer’s borreliosis, all participating researchers seemed to agree, based on the evidence, that persisting low levels of Bb spirochetes account for a relatively greater immune response than other types of infections, in some patients.  This was in agreement with data from decades earlier on human spirochetal infections.  In 1976, in “The Biology of Parasitic Spirochetes”, edited by Russell C. Johnson, it was mentioned that future development of more effective antimicrobials would address the problem of relapsing borreliosis and persisting syphilis.  In that text, before the discovery by Dr. Burgdorfer, Jay Sanford, MD, of Uniformed Services University Medical School, Bethesda, Maryland, stated that: 

 “There are other aspects of the treatment of the relapsing fever, syphilis, and leptospirosis that illustrate similarities and from which therapeutic principles may be developed.  The ability of borrelia, especially the tick-borne strains, to persist in the eye and brain during remission after treatment with arsenic or with penicillin or even after apparent cure is well known.  The persistence of treponemes after treatment of syphilis is a major area which currently requires additional study [references].” 

In the 1970 comprehensive study of louse-borne relapsing fever, published by Bryceson in Quarterly Journal of Medicine, treatment of the borreliosis did not prevent relapse.   

Patients remain infected.  Some are symptomatic and some are not.  This was well-established in the scientific literature before Polly Murray discovered the syndrome that appeared to be of infectious etiology, and appeared to be associated with an arthritis in Lyme, Connecticut.

 Raymond Dattwyler of SUNY Stony Brook found that oral treatment failed as often as 50% of the time in patients.  At that time, Dr. Dattwyler made the suggestion that an intravenous antibiotic with greater CNS penetration, such a ceftriaxone, would be necessary for this CNS infection.  Ceftriaxone worked best, but still some patients did not recover completely or relapsed.

  Treatment Failed Again.

 It is important to point out the recent work of Mark Klempner, MD, Tufts, who found that treating patients with 30 days of ceftriaxone did not appear alleviate symptoms more in the drug-treated patients, than in the patients who received placebo.  That study was seriously flawed.  The FIQ was never validated for Lyme disease.  The attempt at validation showed that Fibromyalgia and Lyme disease were two different disorders.  Klempner’s “Results” must be given only marginal consideration, but within that margin remains the question, what might work better, faster, to bring patients greater symptom relief than the current standard of care, which was used in this trial?   

Among the several important findings that Dr. Klempner did not report in the July 12, 2001, NEJM- the other data he collected- does hold promise.  Just as there is a genetic correlate with treatment-resistant Lyme arthritis, Dr. Klempner found a genetic correlate with Chronic Lyme disease of the seronegative kind- the kind without arthritis as the dominant feature: HLA-DQB1*0602.  Unfortunately, this information was not published such that other researchers might pursue this autoimmune correlate. 

Roland Martin, of the NIH Multiple Sclerosis study group, had previously spent many years studying neuroborreliosis in Germany.  There, he discovered there might be some T cell autoimmunity in neuroborreliosis patients.  Now Dr. Martin studies T cell autoimmunity in MS in the US.  Dr. Klempner referenced Roland Martin’s autoimmune-neuroborreliosis work in:  Is it thee or me? --autoimmunity in Lyme disease, (Nat Med. 1999 Dec;5(12):1346-7)  in which he mentions possible cross reactivity of OspA with myelin, as well as two other potential cross reactive antigens from borrelia with nerve cells.  That there were several hundred neurological adverse events associated with rOspA vaccine appears to give some credibility to this process.  (See www.lyme.org, The Lyme Disease Foundation).

 Although autoimmunity through molecular mimicry is difficult to prove in vivo, the two primary haplotypes associated with MS are HLA-DQB1*0602 and HLA-DRB1*1501.  Roland Martin’s Lyme neuroborreliosis patient from which he extracted potentially autoreactive T cells to human nerve tissue had *1501.  Klempner seems to have identified the other haplotype in Lyme borreliosis patients, who had been physician-diagnosed EM- exposure to Borrelia burgdorferi, but who were seronegative according to the CDC’s standard (personal communication with Dr. Klempner).  That is, some may have had some specific bands on a Western Blot, but not 5 of 10. 

Klempner’s retreatment study resulted in failure to demonstrate adequacy of 30 days of ceftriaxone, which is the standard of care for late Lyme.  Doxycycline for Lyme disease that has invaded the central nervous system was not validated.  The study of the comparative efficacy of ceftriaxone and doxycycline was specifically for non-CNS Lyme disease (3).  Klempner studied the cognitive aspects of borreliosis and found neuropsychiatric deficits, referencing Weinstein’s work.  So that there was a neuropsychiatric component to Chronic Lyme patients’ problems, means Klempner should have known doxycycline was not appropriate for this trial.  It would have better served the public if Klempner reported the positive pathological findings in these patients, since this was not a long term treatment study, but only a trial of the efficacy of the current standard of care.  We might not consider it a complete waste of 4.7 million dollars if we could see the real data. 

Scientific Integrity and Medical Ethics Failed. 

The current standard of care “fails” often, and is the reason why there is resistance by insurance companies to pay for continued care.  The reinterpretation of Klempner’s “results” to the lay media was left to individuals who are often paid by insurance companies to perform independent evaluations in which a judgment is made regarding the diagnosis as well as the treatment, based on records, and not the presence of the patient in what the CDC calls a clinical diagnosis.  Some of these individuals are hired as expert witnesses for insurance companies who wish to deny payment for Lyme disease treatment claims in court cases.  This latest round of reinterpretation for lay-media consumption echoed previous insurance company-supported interpretations of the dynamic of chronic Lyme:   

Lyme patients are not sick, it is the anxiety over the possibility of having Lyme disease that brings people into a medical office.

It’s not Lyme, it’s your divorce.  

Antibiotics are poison.  

Women and girls should not be given the diagnosis of Lyme disease because of their unnecessary use of healthcare resources.    

Perpetuating the notion that there is a chronic Lyme disease incurs the consideration of Munchausens’ or Munchausens by Proxy.  

I call it Lyme paranoia.   

Klempner allegedly found no evidence of Bb DNA in the cerebrospinal fluid of patients that he screened out and claimed ineligible for his study of treatment efficacy initially.  There was at least one patient, who was rejected by Klempner for eligibility, due to the presence of Bb DNA in her CSF.  Therefore, we have reason to believe there may have been others.  Low, or no Bb DNA in the CSF of neuroborreliosis patients is a common finding, and according to Jorge Benach of Stony Brook, may be because the spirochetes are binding to glial cells rather than are free-floating in fluid (the flagellum is on the inside, not the outside).  Klempner then resampled the 129 DNA-negative patients, for a total of 740 samples.  The alleged reason he re-performed this analysis was to look for spirochetal fragments, perhaps released via the Jarisch-Herxheimer reaction, but this was not mentioned in the July 12, 2001 NEJM.  Instead, this information was presented as if it were an assessment of the degree of the negative finding. 

More important than this negative finding, which was to be expected since this was fluid and not tissue, is what he did find and reported previously, matrix-metalloproteinases (MMPs) in the cerebrospinal fluid.  MMPs are recognized as a neurodegenerative process marker in other neurodegenerative diseases.  We know little else about what Klempner found with the 4.7 million dollar grant, because not too much, apparently, was reported.  What we do know regarding other signs of illness process in borreliosis patients was later discussed by Dr. Klempner, verbally, in medical conferences. 

Previously, Klempner found that Bb cultured with human foreskin fibroblasts, resulted in either intracellular compartmentalization (and/or embedding in fibroblast outer membrane) protection of the spirochetes and their subsequent survival after exposure to ceftriaxone for 14 days.  This is consistent with what has known about spirochetal infections of all kinds in all mammals, for decades.  An additional mechanism of spirochetal survival against adverse conditions, the spheroplast, is a well-documented survival mechanism used by free-living spirochetes, as well.  In vitro reports of efficacies of antimicrobials based on the formation of the Bb spheroplast as the “end stage” are therefore invalid. 

There is reason to believe there is a genetic tendency to be more immune-reactive to Borrelia burgdorferi infection of the CNS, just as there is proposed in treatment-resistant Lyme arthritis.  Klempner allegedly found it.  He did not report it.   

There are many other markers of pathological processes that can be identified in significant populations of Lyme borreliosis patients.  Yet, the discovery of these has not led to the development of any formal treatment modality to address them.  MS is a chronic illness for which the symptoms/effects are treated without knowing the cause, as is Lupus and ALS.  “Symptoms” is fairly synonymous with pathophysiological process signs.  It is generally accepted that persons with these other chronic illnesses, with their chronic process signs, are not “treated” once, and then told to please be on their way to a psychiatrist to address their abnormal emotional need to have an illness.  They are treated for their illness. 

Lyme is the one (political) illness in which there has been no formal acknowledgement of these collective chronic process signs in response to low levels of persisting spirochetes, much less, in a comprehensive way.  There has only been controversy over antibiotic “failure”.  Throughout this collection are interspersed examples of how Lyme disease has been spun, so that the reader will have a clearer picture of what patients and Lyme-literate physicians are up against, politically and socially.  As was seen in the first public hearing in Rhode Island April 8 this year, this makes a terrible illness far worse. 

Where do we place the fulcrum now, to effect success?

 THE SCIENTIFIC METHOD

1.       Observation and description of a phenomenon or group of phenomena.

2.      Formulation of an hypothesis to explain the phenomena. In physics, the hypothesis often takes the form of a causal mechanism or a mathematical relation.

3.      Use of the hypothesis to predict the existence of other phenomena, or to predict quantitatively the results of new observations.

4.      Performance of experimental tests of the predictions by several independent experimenters and properly performed experiments. 

The Scientific Method is the reverse of Evidence-Based Medicine.  Evidence-Based Medicine is where experiments are designed to effect the intended limited treatment outcome.  The prototype for this Reversed Scientific Method in practice would be the Klempner 4.7 million dollar CLD “long term” treatment trial.  

Back to basics: What does the data tell us?

 One possible consideration for the Rhode Island Legislature is to simply address that Chronic Lyme disease is just that: a chronic illness.  That then opens the door to new treatments, perhaps symptom-based, that are not antimicrobials, in addition to antimicrobials.  At present, antibiotics are the primary treatment of Chronic Lyme, because of political impediments to advancing discovery of markers of illness processes and addressing them.  If Steere found a genetic link to treatment-resistant Lyme arthritis, and Klempner found a genetic link to seronegative Chronic Neurologic Lyme, then there is a genetic link to treatment- resistant something, and it is not true that Chronic Lyme is a non-entity.  Treatment-resistant Lyme arthritis is not called “Post-Lyme Arthritis Syndrome”.

 The definition of Lyme disease was falsely narrowed to force an acceptable vaccine outcome.  The vaccine was not recommended for persons with active Lyme arthritis.  We’ve all now seen how a false standard failed in practice, with a commercial product: LymeRIX was pulled off the market due to adverse events in at-risk populations.

 It isn’t possible to completely summarize and index what are the pathophysiological signs in burgdorferi borreliosis, with and without coinfections.  These are, formally, “Emerging Infections”.  From one region to another, from one habitat to another, it will never be possible to categorize, with any certainty, which infections should be tested for upon tick bite.  However, starting points in patient management would be to 1) point out the problems associated with the present standard of serodiagnosis, and 2) summarize some of the analyses that have been performed in the past, in markers of pathology, associated with borreliosis.  Establishment of protocols for identifying associated pathological features, regardless of the probability of ever identifying all tick borne infections in any one patient, may at least yield the identification of subsets of patients who have signs of pathology which suggest potential improvement with a pharmacotherapeutic modality other than antibiotics.

 A management plan would be based in making changes to the management of these infections and the management of the physiological changes objectively associated with subjective symptoms, thereby improving patients’ potential for the greatest degree of recovery from symptoms.  All observations re-reported here, although incomplete, are intended to increase that potential.

 ----------

 USE OF SERODIAGNOSIS TOOLS & DETERMINING INFECTION PREVALENCE

It is probable that RI has the highest US infection rate of borreliosis, although the CDC case reported numbers don’t reflect this.  The reasons for this are that:

1) some areas of RI may have the highest tick density in the world, according to Tom Mather at the “Diseases of Summer Conference” at South County Hospital, RI, 2000, and

2) at least in South County, the primary diagnostic lab, Imugen, Norwood, MA., uses none of the CDC’s Dearborn Conference recommended strains of Borrelia burgdorferi, and Imugen’s accuracy performance against this CDC criteria was the lowest reported performance at the Conference of all the labs that participated, 14% accurate (4).

3) CDC criteria in practice, finds Lyme disease 14-22% of the time.  In addition, the disease is at least 10 times underreported (5) due to physicians neglecting to make the effort to fill out the report forms.  Using Imugen testing method and materials puts the identification of potential cases below what it should be, given the reluctance of physicians to report 5 of 10 bands to the CDC.

WESTERN BLOT

 Reporting forms and use of standard strains from all laboratories performing serodiagnosis of Lyme disease in the State of Rhode Island should be reviewed and approved by this Commission before being recommended by the State as a competent evaluating laboratory, effective immediately.   

Laboratories with Western Blot reporting forms which state that “< 5 bands is Normal” must remove this statement from their report forms immediately.  “Less than 5 bands” is not “normal”.  This adds to the diagnostic confusion that has resulted in the extensive misdiagnosis of borreliosis patients in RI. 

Laboratories that report that 5 IgG CDC Dressler/Steere bands means past infection and is not a treatable case must also remove this statement from their forms.  Patients who present with characteristic symptoms of Lyme disease, the criteria for diagnosis as stated by the CDC, would not be having their blood tested if they and their physician did not feel there may be a serologically discoverable illness which required treatment. 

EXHIBIT:  Imugen Report Form 

The 5 of 10 band IgG Dressler/Steere CDC case reporting criteria was based on a calculation (6) and does not represent the empirical observation of what occurs in a typical infection in non-arthritis prone individuals (65-70% of the population).

The outer lanes on the neuroborreliosis blots are standards.  Graphics from:

http://alpha1.mpk.med.uni-muenchen.de/bak/nrz-borrelia/miq-lyme/Frame-MiQ-microbiological53.html

 MiQ Lyme Borreliose, 12 2000, 5. Microbiological diagnosis

Dressler/Steere CDC IgG criteria more closely approximates the antibody concentration found in the arthritis-prone individuals, or those with the HLA-DR4 and DR2 haplotypes associated with Lyme arthritis and antibodies to OspA and OspB, as reported by Allen Steere (7). 

 The antibody concentration response difference between an arthritis-prone individual and a neuroborreliosis-prone individual could be as high as 4-5 times (8).  Where labs report “< 5 bands is Normal”, patients are being misdiagnosed, which is a medical negligence liability. 

At the CDC’s Dearborn Serodiagnosis Conference, October 1994, some labs qualified the Dressler/Steere IgG criteria in the field and only found borreliosis patients responding with 5 of the Dressler/Steere antigens 14 to 22% of the time (4,9).  The CDC’s case reporting criteria is known to be only 30% accurate, by CDC’s own admission (10).  This happens to match the prevalence of HLA-DR4, or the haplotype associated with the high antibody expression in resistant Lyme arthritis- 30- 35%. 

IgG and IgM  

Approximately 30 antigens of Borrelia burgdorferi can be detected via antibody determination; not all are specific to Bb, but in the presence of other Bb specific antigens, some of these markers may be associated with a resultant neuroautoimmune disorder, such as the heat shock proteins, glycolipids, and phospholipids.  That is, more than just outer surface lipoproteins, flagellin, and the few antigens of unknown function, such as P30, identified with Dearborn IgG criteria, are immunogenic.  Other diagnostic markers in pathological response must be considered for diagnosis and addressed in patients because of suspected cross-reactivity. 

With rOspA vaccine now off the market, of course, OspA (31 kD) and OspB (34 kD) can be put back into the serodiagnostic criteria and there will be no use for an OspA-less strain used in Western Blotting, such as what Imugen has been using for the past two years. 

 Before using an OspA-less strain, Imugen was diagnosing antibodies to US Bb strain G39/40, and a strain from Germany.  At the Dearborn Conference, the CDC’s Barbara Johnson recommended not using G39/40, because of insufficient antigen expression.  Oddly, this was the strain, from which Dressler/Steere, the CDC’s current reference for IgG positive antibody criteria, was derived. 

 In addition to using a non-recommended strain, Imugen was using strain FRG (Federal Republic of Germany) to test US patients until they dropped it for the OspA-less strain.  It would seem prudent to that US patients be tested with local US strains, due to strain variation and antigenic variation from environmental pressures (host species range and selection).  A non-systematic review of RI patients’ blot reports from Imugen showed that in every patient (except one who was known to have been infected in Germany), there are fewer positive bands generated from the German strain than the G39/40.  Low passage 2591 is best, due to its higher expression of OspC than B31, or 297, the other two of the three Dearborn recommended strains.  OspC is associated with neurotropism (11, 12, 13) and is thought to be the dominant antigen in a mammalian host (37C), switching from OspA dominance in the tick, with exposure to warm blood.  Neuroborreliosis is the more disabling, and the more common treatment-resistant Lyme illness, therefore using a low OspC-expressing strain would not be helpful.

 OspA and OspB are encoded on same plasmid.  Multiple in vitro cultures, or high-passage without exposure to a mammalian host, results in plasmid loss; loss of plasmid encoded infectivity and virulence.  Strains that have dropped plasmids are not only, therefore, not rare, but demonstrating low in vitro passage is still a requirement for laboratory performance criteria.  The strain neurovirulent strain N40 was developed by passing back and forth between mouse brains and in vitro.  It wouldn’t be hard to imagine developing a strain with mouse joint affinity (G39/40?). 

Two other specific antigens may be added to the Dressler/Steere 10, in addition to OspA and Osp B: P37 (4, 14) and 35kD, a portion of which went into the “C6 ELISA” (15).

 It was well known before the 1980s that the borreliae behave immunologically like the trypanosomes, and this was noted by Allen Steere in 1986 (16, 17).  This is the reason to expand the Western Blot IgM and IgG reporting, and why the LymeRIX vaccine was not qualified properly.  Dressler/Steere IgG, the criteria for CDC case reporting and qualifying the vaccine, is characteristic of the 30-35% of the population who are Lyme arthritis-presenters- the HLA-DR4 types.   

Why these arthritis patients continue to present with the same antigen-stimulated antibodies is unknown, although genotype-associated binding affinities of HLA antigen-presenting molecules and antigen have also been associated in a negative way.  Patients with HLA-DQ1*0602 tend not to have Type I diabetes.  One theory is that whatever is the antigen from a possible viral infection that initiates autoimmune diabetes, simply flops out of this *0602 presenting molecule and doesn’t continue to stimulate.  Donald Wiley (Harvard- now deceased) attempted to show how myelin could “fit” into the typical class II antigen-presenting molecules of MS patients.

 Some believe a chronic infection in a knee joint is due to persisting organisms, which is what Allen Steere discovered by repeated DNA sampling of the synovium in chronic Lyme arthritis patients in 1994.  Bb DNA was found in the synovium for up to 7 years, the time limit of the study, in one patient.  The CDC’s IgM criteria were of Engstrom.  These included OspC, 41, or 39.  SmithKline Beecham also agreed this continued infection might be the possible reason for treatment-unresponsive Lyme arthritis, rather than being autoimmune driven, during the January 31, 2001 FDA vaccine meeting.  This is worth mentioning, because one may then imply from Steere’s work and the vaccine manufacturer’s, that Bb persists in neuroborreliosis.

 Expanding or changing IgM antibodies implies persisting infection and always did, since the Burgdorfer spirochete has similarities in pathogenesis to trypanosomes and relapsing fever borreliae.  Steere reported recently that the continued presence of IgM over time was not associated with a disease state, which is consistent with the concept of asymptomatic infection.  The presence of IgM in asymptomatic infection does not make the presence of IgM antibodies long after the illness, not a marker of illness (17).  Prior to that, Steere perceived the persistence of IgM implied the persistence of the infection (18).  The criteria of Engstrom for IgM are inadequate for diagnosis at any stage of the illness.  Any single specific band, in the presence of symptoms, is as diagnostic as its assigned specificity. 

Not everyone who is infected is symptomatic.  Steere found in 1986 that the ratio of infected asymptomatics to infected symptomatics was 1:1 (19).  The problem associated with that came to the surface during the vaccine trial, when the vaccine was “effective at preventing asymptomatic infection.”  Many people have this observed this differently- the vaccine seemed to be finding the asymptomatic previously exposed patients, and making them symptomatic.  There were 11% more “Unconfirmed Lyme” in the vaccine group, than the placebo group the first year of the trial.  Once the vaccine was on the market, it appeared that mostly patients who had known they Lyme disease before, and had improved, were becoming ill again after receiving the vaccine.  The second year of the trial, the numbers were equal.  Again, only the HLA-DR4 presenters had a 72% chance of being CDC seropositive in IgG (6).   

In empirical exercise, in the field, excluding what Imugen found (14%), CDC IgG criteria of Dressler/Steere was accurate 22% of the time.  The vaccine was 78% effective.  Clearly, those data balance out nicely.  If CDC came up with a standard that was only 10% accurate for a random sampling of all human HLA Class II genotypes, perhaps the vaccine would have been marketed as 90% effective.  There would probably also have been an even greater shift in the increase of “Unconfirmed Lyme” (less than 5 bands) in the vaccine group over the placebo group, than the 11% demonstrated, and reported, in the LymeRIX trial results. 

For every infected symptomatic patient, another remains infected but asymptomatic.  Whether asymptomatic cases should be treated, is controversial, and likely to not be entertained by this Commission, given the magnitude of numbers of patients who are currently very ill and not being treated in any way.  However, whether this represents a risk for reactivation due to life stresses such as surgery, accident, or vaccination, should remain a consideration and patients and physicians should be thus advised.   

Continued careful review of data on immune dysregulation caused by the burgdorferi lipoproteins, especially as concerns intentions for another Osp-based vaccine trial will be necessary.  Another vaccine trial should not go forward until the CDC’s serodiagnostic standard reflects acceptable accuracy of >95% across all human Class II genotypes represented in the US.

 Therefore: 

IgM or IgG should be one of more of at least 14.  The percent specificities of these bands average in the 90s.  The 41kD flagellar antigen is non-specific, but more specific in the absence of periodontal disease or syphilis. 

18 kDa,  21 kDa (OspC) *,  28 kDa,  30 kDa,  31kDa (OspA),  34kDa (OspB),  35 kDa,   37kDa,  39 kDa (BmpA),  41 kDa (Fla),  45 kDa,  58 kDa (not GroEL),  66 kDa, and  93 kDa.

 In addition to these:

55kD: 

Feng S, Barthold SW, Telford SR 3rd, Fikrig E., P55, an immunogenic but nonprotective 55-kilodalton Borrelia burgdorferi protein in murine Lyme disease., Infect Immun. 1996 Jan;64(1):363-5. PMID: 8557366 

Feng S, Das S, Lam T, Flavell RA, Fikrig E., A 55-kilodalton antigen encoded by a gene on a Borrelia burgdorferi 49-kilobase plasmid is recognized by antibodies in sera from patients with Lyme disease. Infect Immun. 1995 Sep;63(9):3459-66. PMID: 7642278 

3-5kD Glycolipid

Oschmann P, Wellensiek HJ, Zhong W, Dorndorf W, Pflughaupt KW. 

Relationship between the Borrelia burgdorferi specific immune response and different stages and syndromes in neuroborreliosis.  Infection. 1997 Sep-Oct;25(5):292-7. PMID: 9334864

“Antibodies against certain proteins and the glycolipid of B. burgdorferi seem to have a prognostic value as to the development of more severe disease or transition to stage III.”

Honarvar N, Schaible UE, Galanos C, Wallich R, Simon MM., A 14,000 MW lipoprotein and a glycolipid-like structure of Borrelia burgdorferi induce proliferation and immunoglobulin production in mouse B cells at high frequencies., Immunology. 1994 Jul;82(3):389-96., PMID: 7959873

Eiffert H, Lotter H, Jarecki-Khan K, Thomssen R., Identification of an immunoreactive non-proteinaleous component in Borrelia burgdorferi., Med Microbiol Immunol (Berl). 1991;180(5):229-37. PMID: 1722277

14kD:

Honarvar N, Schaible UE, Galanos C, Wallich R, Simon MM., A 14,000 MW lipoprotein and a glycolipid-like structure of Borrelia burgdorferi induce proliferation and immunoglobulin production in mouse B cells at high frequencies., Immunology. 1994 Jul;82(3):389-96., PMID: 7959873

OspE is ~19kD

OspF is ~29kD

OspD is ~28kD, Fikrig, Yale University.   

These are all surface exposed lipoproteins associated with virulence, but because of their proximity to other antigens in migration through a Western Blot, they cannot be detected and are less useful.  It was necessary to have a vaccine trial, therefore, progress in serodiagnosis stagnated.  Not all borrelial spirochete strains and species will have exact analogs to these molecules and therefore local (RI) strain sonicates must accompany CDC strains in blot preparation.  Better tests than the Western Blot are 2 dimensional blots and antigen-antibody decomplexing before assay.  However, the gel-based electrophoresis-transfer method could stand development to increase sensitivity and separation, using perhaps capillary electrophoresis (CE).

CE methods and instrumentation are capable of assaying immune complexes.

 There are yet many other antigens, some of which are not lipoproteins (10-, 13-, 33-, 57-, 94- kD etc).

 To look at the problems with serodiagnosis another way, according to Alan Barbour in:

 Antigenic Variation in Vector-Borne Pathogens, Emerging Infections, a CDC pub, Vol 6, No. 5

Alan G. Barbour* and Blanca I. Restrepo, *University of California Irvine, Irvine, California; and †Corporación para Investigaciones Biológicas, Medellín, Colombia

 “Several pathogens of humans and domestic animals depend on hematophagous arthropods to transmit them from one vertebrate reservoir host to another and maintain them in an environment. These pathogens use antigenic variation to prolong their circulation in the blood and thus increase the likelihood of transmission. By convergent evolution, bacterial and protozoal vector-borne pathogens have acquired similar genetic mechanisms for successful antigenic variation. Borrelia spp. and Anaplasma marginale (among bacteria) and African trypanosomes, Plasmodium falciparum, and Babesia bovis (among parasites) are examples of pathogens using these mechanisms. Antigenic variation poses a challenge in the development of vaccines against vector-borne pathogens.” 

It therefore also poses a challenge in serodiagnosis.  At least an attempt to find local strains to test local patients using better separation methods with greater sensitivity is clearly the direction to start in, to identify Lyme patients so that they can be treated appropriately.  It most certainly doesn’t help to be testing RI patients with a strain from West Germany, as Imugen did for several years (personal communication with Victor Berardi, Imugen).  That the CDC remained stagnant on this issue was because they sponsored the serodiagnostic criteria for the vaccine manufacturers.  The CDC therefore had to continue to support their criteria.  With the vaccine off the market, CDC no longer has to support the Dressler/Steere and Engstrom criteria. 

Coinfection with at least an Erhlichia results in a diminution of antibody response.  Ticks in CT are infected with an Erhlichia 50% of the time (Magnarelli, NIH Rare Diseases Conference, 1996).  Coinfections can be the reason for seronegativity and the reason patients should be routinely tested for coinfections when Lyme is suspected. 

Local strains must be employed for testing local patients.  Identifying local strains for serodiagnosis in RI is a project for which the State should request NIH funding. 

 The goal for a RI Public Health TBDs management program is to identify Bb infected patients and not to narrow disease definitions to qualify patented biological methods and materials, such as what occurred with the Wampole PreVue ELISA, the C6 ELISA, or the LymeRIX vaccine. 

 ELISAs

 A screening test, such as a Lyme ELISA, is completely unacceptable to identify human cases of Lyme borreliosis, at the recommended concentrations of test kit antigens (20) and serum dilution. 

Patients who are Western Blot positive are often ELISA negative (21).  All false positives ever claimed to occur in Lyme borreliosis, have been identified via ELISA.  False positive ELISAs allegedly occur in Rheumatoid arthritis, Lupus and syphilis (22).  It is impossible to have a false positive with a Western Blot, since a specific band is a specific band, and specificity assignments are high with the CDC’s chosen antigens for US patients.  That a person may have a negative ELISA and still have many Bb specific bands via Western Blot, demonstrates how much less sensitive the ELISA is, than is claimed.  It is claimed to be more sensitive than a Western Blot, but less accurate.  It is less sensitive than a blot and therefore is the exact opposite of a screening test. 

The newest ELISA, yet to be on the market, will include several partial-recombinant OspCs, and similar specific antigens, so it will be more specific than flagellin-based ELISAs (20).  The concept of this new ELISA is acceptable, but if this test is used, it seems that a trial of doubling the sample concentration may make up for current lack of sensitivity.  Hopefully someone will have the funding to compare this new ELISA, with adequate dilution, to a Western Blot from a low passage 2591 strain.  In lieu of the possible qualification of this new ELISA, as described above, the ELISA should be dropped altogether as a screening test.  Screening tests are acceptable for livestock, not people.

 LYME-LIKE ILLNESS

 Not all Bb antigens are detectable by Western Blot, and borreliae other than burgdorferi are in the continental United States and cause a Lyme-like illness.  B. lonestari is transmitted via the Lone Star tick and Master’s disease is a Lyme-like illness transferred by the Lone Star tick.  There is fair diversity of borreliae in the US that is non-sensu stricto.  It hasn’t been ruled out that they cause an illness similar to relapsing fever or Lyme borreliosis, and neither has there been any attempt by the CDC to change serodiagnostics to reflect these new discoveries.  Of 60 or more Bb sensu lato strains and several newly discovered species of borreliae found in Lyme endemic areas, “Lyme disease” so far, can only be detected by a few of the antigens from 3 strains of Bb sensu stricto (23).  If persons are infected by non-sensu stricto strains, or non-burgdorferi species, the CDC currently has no design to recognize them. 

 The following abstract pertains to RI, as well as NY and CT:

 Glen A. Scoles; Michele Papero; Lorenza Beati; Durland Fish

Source: Vector Borne and Zoonotic Diseases      Volume: 1 Number: 1 Page: 21 -- 34

DOI: 10.1089/153036601750137624  Publisher: Mary Ann Liebert, Inc.

 “Abstract: A species of Borrelia spirochetes previously unknown from North America has been found to be transmitted by Ixodes scapularis ticks. Infected ticks are positive for Borrelia spp. by DFA test but negative for Borrelia burgdorferi by polymerase chain reaction (PCR) using species-specific primers for 16S rDNA, outer surface protein A, outer surface protein C, and flagellin genes. A 1,347-bp portion of 16S rDNA was amplified from a pool of infected nymphs, sequenced, and compared with the homologous fragment from 26 other species of Borrelia. The analysis showed 4.6% pairwise difference from B. burgdorferi, with the closest relative being Borrelia miyamotoi (99.3% similarity) reported from Ixodes persulcatus in Japan. Phylogenetic analysis showed the unknown Borrelia to cluster with relapsing fever group spirochetes rather than with Lyme disease spirochetes. A 764-bp fragment of the flagellin gene was also compared with the homologous fragment from 24 other Borrelia species. The flagellin sequence of B. burgdorferi was 19.5% different from the unknown Borrelia and showed 98.6% similarity with B. miyamotoi. A pair of PCR primers specifically designed to amplify a 219-bp fragment of the flagellin gene from this spirochete was used to survey field-collected I. scapularis nymphs from five northeastern states (Connecticut, Rhode Island, New York, New Jersey, and Maryland). Positive results were obtained in 1.9-2.5% of 712 nymphs sampled from four states but in none of 162 ticks collected from Maryland.  Transovarial transmission was demonstrated by PCR of larval progeny from infected females with filial infection rates ranging from 6% to 73%. Transstadial passage occurred from larvae through adults. Vertebrate infection was demonstrated by feeding infected nymphs on Peromyscus leucopus mice and recovering the organism from uninfected xenodiagnostic larvae fed 7-21 days later. Considering the frequency of contact between I. scapularis and humans, further work is needed to determine the potential public health significance of yet another zoonotic agent transmitted by this tick species.”

 As mentioned in the previous section, some coinfections inhibit antibody response. 

Pachner AR, et al, Detection of active infection in nonhuman primates with Lyme neuroborreliosis: comparison of PCR, culture, and a bioassay. J. Clin Microbiol. 1998 Nov;36(11):3243-7. PMID: 9774573:

 “The presence of specific anti-B. burgdorferi antibody in the CSF is the most widely used assay for Lyme neuroborreliosis. In the immunocompetent NHPs in our study it was a very successful assay for detection of CNS invasion. However, it is frequently false negative, especially early in the course of the infection or if there is transient immunosuppression. Transient suppression of the anti-B. burgdorferi immune response in humans could occur in instances of coinfection, i.e., simultaneous transmission via the tick of pathogen other than B. burgdorferi. Coinfection of ixodid ticks has been demonstrated for a number of pathogens, including the agent of human granulocytic ehrlichiosis (16), babesiosis (25), and tick-borne encephalitis virus, as well as for a newly described virus (23) and bacterium (22). In a recent study from an endemic area of New Jersey, 18% of infected ticks were infected with more than one readily identifiable pathogen (25); this number is likely an underestimate, since many tick-borne agents have not yet been identified. Infections, even subclinical ones, with a variety of pathogens have been demonstrated to suppress the expected host immune response (2). Thus, mild immunosuppression as accomplished in this study was designed to mimic conditions in the human host which allow B. burgdorferi in the natural state to gain a firm foothold in the CNS in the 10 to 15% of B. burgdorferi-infected patients who develop clinically symptomatic nervous system disease.”

22. Schwarzova, K., and I. Ciznar. 1996. Spirochetal non-Borrelia microorganisms isolated from Ixodes ricinus. Folia Microbiol. 41:175-180.

 The bioassay used in the study above was the mouse infectivity test, such as is used in syphilis.  This is a better method than PCR, since PCR can be limited by inadequate primers. 

There appears to be intent to use what is left over of rOspA (LymeRIX) to sterilize ticks with Bb ss OspA-bearing spirochetes in their gut, via adding rOspA to some kind of wild mouse food.  We don’t know what effect this might have on shifting the spirochete populations away from those expressing antigen with which we are are familiar.  Getting rid of ticks is more important that getting rid of Bb ss, because of the known and unknown coinfections.

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SURVEILLANCE, LONG TERM GOALS

             IMPROVE METHODS OF DETECTION OF ILLNESS

            IMPROVE SURVEILLANCE FOR ALL TBDs

 Any new strains and species of Borreliae identified in RI and the three CDC recommended strains of Bb ss, together should be used in Western Blotting in Rhode Island’s own university and hospital labs, as is done in Europe.   

WB is not a complicated procedure and should be performed in all hospital labs until faster, more sensitive antigen/antibody assay, instrumentation and methods, are developed and put into widespread use. 

The health management goal is to be able to identify a borreliosis patient while still in the early, acute, flu-like infection stage, and before borreliae are distributed to the nervous system (“Quantitative immunohistochemical analysis demonstrated infection of spirochetes in kidney interstitium and brain as soon as 2 days postinoculation”- Bergstrom, et al, Infect Immun 2001 Sep;69(9):5832-9).  This means we should target diagnosis turnaround-time of less than 12 hours, i.e., same-day testing and results.  The technology is available, but one cannot put that level of technological sensitivity into a test kit, such as an ELISA test kit.  There has been commercial pressure to keep testing poor, sloppy, slow, and therefore undeniably harmful to TBDs patients.  It is claimed that antibody response is low in early Bb-infected patients, but that hasn’t been proven to only be a function the host’s response.  It may be a function of method sensitivity or that antibodies require decomplexing before assay. 

Funding will be necessary to accommodate local vector/host low passage antigen facility.

 Since RI is a sentinel state, and as the Massachusetts islands are sentinel islands for CDC’s TBDs surveillance, it is imperative that RI institute measures to identify new TBDs.  Coastal islands and states are monitored in the US and Europe, because many TBDs make their way into new habitats via sea birds (24).

 Several objective scientific methods and evidence for pathology, in addition to specific markers of infection with borrelia in borreliosis patients have been developed and discovered.  These analyses should be routinely employed in diagnosis and differential diagnosis in patients who are both seronegative and seropositive by the RI local (sl and ss; all) and CDC’s Bb (ss) antibody criteria.  Better separation methods besides gel electrophoresis with solid polymer blot substrate, might accommodate these.

 ERHLICHIOSIS- An example of the current state of the art, just with monocytic:

The Division of Microbiology and Infectious Diseases, NIAID with support from the Office of Rare Diseases convened a workshop on "Human Ehrlichiosis" on September 5, 1996.

 Human infections with Ehrlichia chaffeensis: clinical, pathological, and immunologic aspects.
David H. Walker, M.D., Department of Pathology, The University of Texas Medical Branch at Galveston, Galveston, TX 77555-0609.

“The spectrum of syndromes and distribution of severity of human monocytic ehrlichiosis (HME) is incompletely defined. Two prospective active surveillance studies suggest the contradictory views that HME is usually either asymptomatic or requires hospitalization. The largest series reporting 237 passively collected cases includes 62% hospitalized patients, a median duration of illness of 23 days, and a systemic disease often with gastrointestinal, hepatic, neurologic, and hematopoietic involvement. Bone marrow hyperplasia, granulomas, and erythrophagocytosis, multifocal hepatocellular necrosis, perivascular lymphohistiocytic infiltrates, and meningitis are known pathologic lesions. The pathology of HME has yet to be investigated adequately. Pathologic study of fatal cases suggests that HME can occur as an opportunistic infection or conversely can induce immunosuppression.  The spectrum of syndromes and distribution of severity of human monocytic ehrlichiosis (HME) is incompletely defined. Two prospective active surveillance studies suggest the contradictory views that HME is usually either asymptomatic or requires hospitalization. The largest series reporting 237 passively collected cases includes 62% hospitalized patients, a median duration of illness of 23 days, and a systemic disease often with gastrointestinal, hepatic, neurologic, and hematopoietic involvement.  Bone marrow hyperplasia, granulomas, and erythrophagocytosis, multifocal hepatocellular necrosis, perivascular lymphohistiocytic infiltrates, and meningitis are known pathologic lesions. The pathology of HME has yet to be investigated adequately. Pathologic study of fatal cases suggests that HME can occur as an opportunistic infection or conversely can induce immunosuppression.  A serious flaw in our current state of knowledge is the laboratory basis for the diagnosis of HME, which is overwhelmingly serologic.  A small fraction of cases have been documented by Ehrlichia chaffeensis- specific PCR. Only three isolates of E. chaffeensis have been established, all from cases of human illness. Each isolate differs from the others genetically and antigenically. Ehrlichia canis was isolated from a healthy seropositive person. It is quite likely that all of the Ehrlichia species capable of causing human infection have not yet been discovered. The genetic and antigenic diversity of E. chaffeensis itself s incompletely known as are their potential strain-determined pathogenicity. The ehrlichical virulence factors have yet to be identified as well as the host factors that determine host resistance and the severity of illness. Likewise, the mechanisms of immunity, or indeed the existence of protective immunity to E. chaffeensis, have yet to be established. There are serious deficiencies in each of the animal models of ehrlichiosis, particularly for the investigation of immune mechanisms against E. chaffeensis. The problems with the models include distant genetic relationship of E. chaffeensis with the E. risticii-E. sennetsu genogroup, lack of characterization of some models for the target cells and pathologic lesions, unrealistic route of inoculation (certainly not via tick-bite transmission), and lack of quantitation of the ehrlichical inoculum and the time course of the organ infectivity titers. HME poses substantial challenges in diagnosis, pathogenesis, and immunity. Research, optimally collaborative, interactive, and multidisciplinary, holds the answers.”

BABESIOSIS

Persistent parasitemia after acute babesiosis.,  N Engl J Med  1998 Jul 16;339(3):160-5,

Krause PJ, Spielman A, Telford SR 3rd, Sikand VK, McKay K, Christianson D, Pollack RJ, Brassard P, Magera J, Ryan R, Persing DH., 

Department of Pediatrics, Connecticut Children's Medical Center and University of Connecticut School of Medicine, Hartford 06106, USA.

“BACKGROUND: Babesiosis, a zoonosis caused by the protozoan Babesia microti, is usually not treated when the symptoms are mild, because the parasitemia appears to be transient. However, the microscopical methods used to diagnose this infection are insensitive, and 

few infected people have been followed longitudinally. We compared the duration of parasitemia in people who had received specific antibabesial therapy with that in silently infected people who had not been treated. METHODS: Forty-six babesia-infected subjects were identified from 

1991 through 1996 in a prospective, community-based study designed to detect episodes of illness and of seroconversion among the residents of southeastern Connecticut and Block Island, Rhode Island. Subjects with acute babesial illness were monitored every 3 months for up to 27 

months by means of thin blood smears, Bab. microti polymerase-chain-reaction assays, serologic tests, and questionnaires. RESULTS: Babesial DNA persisted in the blood for a mean of 82 days in 24 infected subjects without specific symptoms who received no specific therapy. 

Babesial DNA persisted for 16 days in 22 acutely ill subjects who received clindamycin and quinine therapy (P=0.03), of whom 9 had side effects from the treatment. Among the subjects who did not receive specific therapy, symptoms of babesiosis persisted for a mean of 114 

days in five subjects with babesial DNA present for 3 or more months and for only 15 days in seven others in whom the DNA was detectable for less than 3 months (P<0.05); one subject had recrudescent disease after two years. CONCLUSIONS: When left untreated, silent babesial 

infection may persist for months or even years. Although treatment with clindamycin and quinine reduces the duration of parasitemia, infection may still persist and recrudesce and side effects are common. Improved treatments are needed.”  PMID: 9664092 

The anecdotal evidence is that mortality from babesiosis is high in Rhode Island.  Because RI is a sentinel state, funding should be sought to improve indentification of new variants in persons and in ticks.  University and hospital facilities should coordinate in granted projects for efficient use of available resources.

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 PATIENT MANAGEMENT IN ADDITION TO IMPROVED DETECTION OF LYME EXPOSURE

            METHODS AND MARKERS OF PATHOPHYSIOLOGY

        QUALIFICATION OF PATIENT PRESENTATION

MARKERS OF PATHOPHYSIOLOGY

 The following are some, but not all markers of pathophysiology.  They are noteworthy because they appear to be related to cognitive changes in borreliosis patients.  If Lyme disease was just an arthritis, there would be very little It is not known whether antiphospholipid or antiglycolipid antibodies seen in borreliosis patients are of bacterial origin or the result of neurological damage and are therefore autoantibodies.  The 5 kD glycolipid is specifically a marker of borreliosis and is associated with more severe neurological disease, as are the antiphospholipid cases and is suspected in borreliosis patients with anti-brain antiganglioside antibodies.  Additionally, it has been suggested that possibly autoreactive T cells, matrix-metalloproteinases (MMPs), quinolinic acid (QUIN), neopterin, and glial fibrillary acid protein (GFAp) in the CSF are signs of ongoing neurological injury.  These process-markers are addressed in other illnesses, e.g., clinical trials of MMP inhibitors in MS and, cyclophosphamide for monoclonal gammopathies and for GFAp in Alzheimer’s, glatiramer acetate and neuroborreliosis (which failed).  There are other objectively detectable signs of pathology in borreliosis patients, such as increased CSF protein, pleocytosis, dysregulated cytokines, gadolinium-enhanced MRI imaging which used for meningitis detection, and EEG changes.

 That these process-markers overlap markers of pathophysiology in other illnesses, especially in other illnesses where there is a significant association to Bb exposure, such as in ALS and MS, is the objective evidence that borreliosis is the multi-symptom disease that subjective reporting first showed.  It is not to say that Lyme causes MS or ALS.  It is to say, symptoms reported by borreliosis patients have a basis in reality.  A differential diagnosis is made based on other disease-specific criteria.