Dr. Baker: Our next speaker, this will be a little bit different in that Dr. Volkman is on the telephone I hope. Are you there?

Dr. David Volkman: Yup.

Dr. Baker: David, are you there?

Dr. Volkman: Yup.

Dr. Baker: Okay. So that

Dr. Volkman: Can you hear me?

Dr. Baker: Yes, we can hear you well.

Dr. Volkman: Okay, thank you.

Dr. Baker: And, please begin.

Dr. Volkman: Okay. I would like to thank the IDSA for their kind invitation.

Ticks can transmit many microbes including Borrelia, Babesia, Anaplasma, Rickettsia, and deer-tick encephalitis virus. Ticks spread Borrelia, or Lyme disease has become the most prevalent arthropod-borne disease in North America since its discovery in 1984. I’ll address with evidence-based data what is known and what is still unknown about this.

What we know about Borrelia is summarized here. Borrelia disseminates systemically from its cutaneous injection site to the joints, the central nervous system, and the heart. After the infection, there is evidence that Borrelia persists. Specifically, a tick bite in the spring may

present as an inflamed knee in the fall. Once established, symptoms can be resistant to some antibiotic treatments.

The humeral response to infection depends on several factors and is variable. Other issues that should be reviewed by this panel include the misapplication of the CDC’s surveillance case definition and the flawed prophylactic recommendation made by the old Guidelines Committee.

Persistent Borrelia infection has been observed in both man and animals. In dogs, Borrelia migrates through tissues following tick inoculation, producing episodes of acute arthritis and establishes chronic infection. After dissemination, it can be found by PCR-polymerase chain reaction in connective and CNS tissues. The spirochete can even survive antibiotic treatment in a proportion of the animals.

A similar situation occurs in mice. If infection is untreated for four months, it becomes difficult to eradicate. Ten to twenty percent of mice have residual Borrelia even after extensive parenteral antibiotic treatment. If brief and inadequate antibiotic treatment is given at the time of infection, such as a single oral dose of doxycycline which is recommended for prophylaxis by Guidelines Committee, 80% of the mice remain infected.

In man, there is strong PCR evidence that Borrelia disseminates to the joints and CNS during infection. Borrelia DNA is found in knee effusions and CSF. However, the evidence that it persists in man after standard antibiotic treatment is still controversial. One reason is limited tissue samples that are available. In experimental animals, CNS or joint tissue can be harvested, ground up, and extensibly studied for Borrelia. In man, this is of course not feasible.

As many in the panel know, Treponema is a spirochete similar to Borrelia which disseminates to the CNS and connective tissue with prolonged infection. Historically, neurosyphilis sometimes proved refractory to the brief courses of antibiotics used for primary infection. However, despite falling antibody titers, CNS gumma were found at necropsy. Thus, longer courses of antibiotics for neurosyphilis were instituted.

As stated, PCR can detect Borrelia DNA in synovial fluid of patients with chronic arthritis. Some patients have Borrelia in their knee effusions even after some antibiotic treatment. Lyme arthritis that persists after antibiotic treatment may be due to persistent resistant spirochete forms long after the patient was first infected. However, we don’t yet have sensitive tools to demonstrate this. PCR can also detect Borrelia DNA in CSF samples in patients with the symptoms of neuroborreliosis.

Several studies have found that after chronic infection and parenteral antibiotics, a small but significant number of patients have some persistent symptoms. Methods for determining if these symptoms are due to persistent Borrelia infection, as occurs in dogs and mice or to immune phenomena as proposed by some, are currently unavailable.

In conclusion, Borrelia can persist in joints and the CNS. In animals, they persist even after parenteral antibiotics. Similar persistence remains controversial in man. An estimated 10% of 40,000 people infected annually may have persistent symptoms. In an analogous situation, neurosyphilis requires extensive antibiotic therapy for its eradication. The optimal therapy for the eradication of disseminated Borrelia has not been determined as yet.

In both man and mouse, once Borrelia infection is established, it's difficult to eradicate even with repeated parenteral antibiotics. Barthold was unable to cure 10-20% of chronically infected mice using sustained parenteral ceftriaxone. Optimal antibiotic regimens for eradicating persistent infection have not yet been determined.

Serological responses to this bacterial infection are well characterized. This figure shows variable antibody responses of patients at different stages of untreated Borrelia infection. EM sera, which were all ELISA negative, have little reactivity. Here they were more intensely stained to visualize the faint bands. Initial Borrelia infection at the EM stage cannot, cannot, be judged by the persistence of anti-Borrelia antibody. However, after six months of untreated infection, much new reactivity becomes apparent -- on the right. Conversely, increased bands stop developing after exposure to antibiotics even if the treatment does not eradicate the remaining Borrelia completely. So judging infection by the number of bands after a patient has

received some therapy is unreasonably restrictive, and infection often cannot be excluded on the basis of serology alone.

The next figure compares a patient’s response to different strains of spirochetes with a human monoclonal anti-Borrelia antibody I developed, called D7, which is reactive with the core of the spirochete. Although there is good cross-reactivity between different burgdorferi species, lanes 1-3 on the right, the intensity of reactivity is diminished when the North American sera is tested against the European strain of Borrelia, lane 3. Little reactivity was seen against Borrelia hermsii or the Treponema, lanes 4-7. The monoclonal on the left binds to all strains of Borrelia, lanes 1-4, but not to the Treponema.

The conceit that we can dictate to the immune system to which antigens it should respond most strongly is incorrect. Although efforts to standardize ELISA assays have been made, their inability to detect some infected individuals has not changed. Claims of 100% sensitivity are falsely based on a selection bias in which ELISA positivity is an inclusion criterion. Newer synthetic antigens have been shown to be slightly less sensitive as antigen targets than whole sonicated Borrelia.

In conclusion, antibodies develop against a range of Borrelia antigens over time. Antibiotic therapy arrests further development of more reactivity once it is given. Borrelia burgdorferi species are highly cross-reactive. Infected patients may have zero to multiple bands of reactivity. If antibiotics are given in the early infection, a negative ELISA or Western blot does not rule out infection. New engineered antigens are no more sensitive than simple sonicated whole Borrelia. Serology detects only 75% of infected people in unbiased studies. A negative serology does not rule out Lyme disease.

The observations that some patients have chronic Borrelia infection but lack any serological response have been questioned. The characteristics of our original 17 patients are shown here. All suffered from either neurological of arthritic signs frequently attributed to chronic Lyme disease. They all had a previous EM, chronic symptoms, early antibiotic exposure, a negative ELISA and Western blot IgG and IgM, and they all had positive T cell responses to Borrelia.

The somewhat counterintuitive observation of seronegative Lyme disease was demonstrated in the New England Journal of Medicine in 1988. The production of anti-Borrelia antibodies is abrogated by removing the bulk of immunizing inoculum early after exposure. This is similar to preventing Rh-negative mothers from developing anti-RH antibodies with a shot of RhoGAM which removes circling Rh-positive fetal cells after delivery. A single dose of oral doxycycline after an infected tick bite does the same thing by removing sufficient bacteria to block both seroconversion and EM in 87% of tick-infected subjects.

This table shows a vigorous T cell response in these 17 selected seronegative patients with signs consistent with chronic Lyme disease.

Despite the difficulty some have had in performing the T cell assay and its cross reactivity with other spirochetes which are shown here, T cell responses remain a reliable indicator of previous Borrelia exposure and is seen in some seronegative patients. Seronegative T cell blastogenesis has been confirmed in other laboratories including that of Steere. The assay itself is cumbersome, slow, and expensive. So it is poorly suited for screening purposes. However, other measures of T cell reactivity may be developed in the future.

Although early antibiotic treatment can block antibody responses, it will not eradicate some infections. Thus, despite the assertions of the Guideline Committee, patients can be chronically infected but seronegative.

Seronegative Lyme disease was confirmed elsewhere by obtaining PCR-positive Borrelia DNA from seronegative subjects with either chronic neuroborreliosis or effusion positive arthritis. In one study, seven of seven seronegative patients with chronic neuroborreliosis were PCR positive in their CSF. Importantly, several other laboratories have detected PCR-positive Borrelia DNA in the CSF or joint fluid of seronegative patients.

In a related study by Luft, a five-day Z-PAK azithromycin was given to volunteers with new EM. Although azithromycin was ineffective in curing borreliosis in half the subjects, the

antibiotic effectively abrogated a serological response. Even though 50% of the subjects developed chronic arthritis or CNS symptoms after six weeks, most remained seronegative.

Seronegative Lyme disease conclusions include: Borrelia specific T cell blastogenesis has been confirmed in both cured and chronic patients. PCR-positive Borrelia DNA has been found in seronegative patients. Chronic Borrelia infection may cause chronic arthritis or CNS symptoms. After established infection, symptoms can resist antibiotic treatment in some people.

In summary, both persistent seropositive and seronegative Lyme disease has been confirmed in chronic patients by PCR-positive Borrelia DNA found in arthritis effusions and CSF.

Two issues which are still confused in the literature are the CDC’s case definition of Lyme disease and the Guidelines recommendation for tick bite prophylaxis.

The CDC surveillance case definition was created in 1989 to track the geographically expanding Lyme disease epidemic. We intentionally defined it restrictively to detect only definitive cases, and we intentionally excluded equivocal ones. The CDC has repeatedly stated that its definition should never, never be used clinically. Despite this oft published warning, failure to meet the surveillance case definition criteria are wrongly equated with not having Lyme disease. Again, although this definition can be used to detect some previously infected individuals, it should never be used to exclude infection of potentially infected people.

The prophylaxis recommendations of the Guidelines Committee should be immediately withdrawn. The Guidelines Committee has recommended a single oral dose of doxycycline to prevent Lyme disease from a tick bite. A similar treatment failed to prevent persistent infection in 80% of mice so treated. Although human studies showed that a single early dose of doxycycline blocks both seroconversion and EM in 87% of bitten subjects, no long-term follow-up was done, and fever and flu-like symptoms were ignored. No measure of chronic Lyme disease was obtained.

Treatment with an ineffective dose of azithromycin as used in one arm of a treatment trial for erythema migrans resulted in chronic symptoms in half the subjects. However, most remained seronegative. The single oral doxycycline regiment may leave recipients with chronic Borrelia infection but no detectable diagnostic antibodies and should be strictly restricted.

Dr. Baker: Dr. Volkman, you have thirty seconds to finish up.

Dr. Volkman: Conclusions are listed here. Persistent borreliosis is a proven infectious disease and should be included in differential diagnoses. Serology can detect previous Lyme disease infections but cannot be used to exclude current infection. The CDC criteria for tracking Borrelia epidemic should not be misapplied to diagnose infection. That was never its intention. And lastly, the prophylaxis recommendation of the Guidelines Committee is ineffective and may leave inadequately treated individuals both chronically infected and seronegative.

Dr. Baker: Thank you very much for your presentation and especially for joining us by phone. We’re quite appreciative of of your testimony. And now, we have some time for questions from the panel?

Dr. Paul Lantos: I have

Dr. Baker: This is Dr. Lantos.

Dr. Paul Lantos: Thank you very much for the presentation. I have two questions for you. First, how do we exclude any case of Lyme disease if we can't rely on serologic testing and we are willing to accept relatively nonspecific symptoms as a sign of Lyme disease? In other words, how do we decide who does not have it?

Dr. Volkman: Okay. That’s obviously a critical concern. Basically, the first thing is to open the open the possibility that we have these, it’s the [recording unintelligible here] serologically. I I can [ ? ] T cell tell you if you’ve been exposed but I can’t tell you if you have it now. So the answer I guess is we need to develop other tests for the presence of [recording unintelligible here.] I can’t answer your question. It's a very good question, we don’t have the answer right now.

Dr. Lantos: Thank you. I have one additional question and that is, your demonstration of PCR-positive positive joint fluid and cerebrospinal fluid reminds me of, I guess it was the mid-nineties, the demonstration of chlamydia in atherosclerotic coronary artery disease lesions that were thought for awhile to be pathogenic but antibiotic therapy was proven to not modify the pathogenesis of atherosclerosis. Do we know if there's a clinical correlate of seronegative PCR positivity in joint fluid?

Dr. Volkman: Joint fluid is difficult because the antibiotic levels in joint fluid are much higher than in the CNS. So I really can't tell you the answer to that. In general, there is a clinical correlation in cases. Dr. Steere has the largest cohort of positive joint fluid so he could better answer the question than I could. In general, I think that he said his position I don’t want to represent his position but he said that chronic Lyme disease does not exist. So in answer to your question, I don't know what's the clinical relation.

Dr. Lantos: Thank you.

Dr. Baker: Dr. Duray?

Dr. Paul Duray: Again, thank you for for this for your work in this very important area. This dispels a lot of false notions that the clinical medicine has been laboring with in several decades. The question is simple and quick. Is the material the material that you’ve summarized for us in your laboratory, is this would this then be sufficient to block out the idea of one- to two-tablet prophylaxis once and for all or should we be more careful?

Dr. Volkman: Dr. Duray, thank you that was a great question upon a point I tried to make. It’s not my lab, it’s the Mass lab of [recording unintellible] Basically, when mice were treated with [recording unintellible] 80% then go on to have persistent [recording unintellible ]. To me that means [single-oral dose doxycycline is] that’s not a very good prophylactic. Okay?

Dr. Duray: Thank you!

Dr. Baker: Thank you, Dr. Volkman. We are now going to break for lunch.

Dr. Volkman: Okay. Thank you for your forbearance.

Dr. Baker: Thank you again.