Ritchie Shoemaker, Dennis House
Center for Research on Biotoxin-Associated Illness (a not-for-profit Corporation)
500 Market Street, Suite 102, Pocomoke City, MD 21851

Introduction. Since the first description of Lyme disease there have been subsets of patients identified with persistent symptoms, refractory to antibiotics. A recent prospective study of 51 Post-Lyme Syndrome (PLS) patients demonstrating a mid-spatial frequency deficit in visual contrast sensitivity (VCS) showed significant reduction of symptoms with use of a cholestyramine (CSM) treatment protocol. The symptoms and VCS deficits in the PLS patients were similar to those demonstrated by patients with other chronic, neurotoxin-mediated illnesses reported previously. Early in the course of CSM treatment, 33% of the 51 PLS patients experienced a significant intensification of symptoms, a phenomenon not seen with different biotoxin exposures. Pretreatment in 12 subsequent PLS patients with pioglitazone, a PPAR agonist, was shown to prevent the intensification associated with CSM use and to reduce plasma levels of a pro-inflammatory cytokine, TNF alpha. A multisite, prospective clinical trial on pioglitazone and CSM use in PLS patients was conducted to confirm benefit of CSM therapy in PLS and determine if pretreatment with pioglitazone could prevent intensification. Methods. All patients referred to the study had long-standing symptoms following a known tick bite or exposure to areas where others had tick bites, and a clinical and/or laboratory diagnosis of Lyme Disease. Diagnostic parameters used by referring physicians included a history of erythema chronicum migrans (ECM) rash, ELISA assay, Borrelia burgdorferi Western blot, LUAT assay, PCR, CSF antibodies to B. burgdorferi and culture. Patients with a clinical diagnosis of PLS but without a positive serologic test were included at the discretion of the attending physician. 241 patients were included in the study at one of two centers, Pocomoke, MD or Chico, CA. All patients had past antibiotic treatment, but retained symptoms refractory to antibiotics. All patients were pretreated with pioglitazone (pio), 45mg daily for 10 days. On day 6 of pio, CSM treatment was initiated. An orally administered checklist monitored symptoms, and sequential VCS testing was performed. CSM was continued until the treatment endpoints of maximum symptom abatement and maximum improvement in VCS scores were noted. No antibiotics or other therapeutic interventions were administered during the study. Results. No patients had adverse effects from pio, including hypoglycemia or abnormal liver function tests. Only 5 of 241 (2%) patients experienced significant symptom intensification, not severe enough to stop treatment. 83.8% of patients had at least 50% reduction of symptoms following use of the CSM protocol, with complete resolution of symptoms in 23%. 3.4% of patients had no improvement. VCS deficits prior to treatment and resolution with treatment were better correlated with symptom abatement than other diagnostic markers. Conclusion. The results support the hypotheses that: 1) PLS may be a chronic, neurotoxin-mediated illness; 2) antibiotic treatment should be followed by adjuvant treatment with the pio and CSM protocol; 3) VCS is an effective diagnostic tool that can indicate neurotoxicity in PLS patients and; 4) TNF may participate in the intensification reaction. These results must be confirmed in a prospective double-blinded, placebo-controlled clinical trial, with frequent monitoring of TNF levels measured appropriately by validated laboratory protocols.

Use of atovaquone (Mepron) in patients coinfected with Borrelia burgdorferi and Babesia microti with symptoms refractory to antibiotics and cholestyramine.

Ritchie Shoemaker, Dennis House
Center for Research on Biotoxin-Associated Illness (a not-for-profit Corporation)
500 Market Street, Suite 102, Pocomoke City, MD 20851

Background

A recent two-site study of 341 patients with Lyme Disease and symptoms refractory to antibiotics (Post-Lyme Syndrome, PLS) showed benefit from a treatment protocol using pioglitazone to lower proinflammatory cytokine levels and cholestyramine (CSM) to bind and eliminate toxins. Statistical analyses showed a significant reduction in the number of symptoms and a significant improvement in visual contrast sensitivity (VCS) scores following treatment. These improvements paralleled those seen in patients with symptoms following exposure to other biotoxin-forming organisms, including dinoflagellate and fungal species. In the PLS studies, however, about 8% of patients did not respond to the toxin-binding protocol. Serologic measures indicated that these patients were disproportionately coinfected with Borrelia burgdorferi and Babesia microti. The coinfected patients had received 3 weeks of atovaquone, targeting b. microti, in combination with azithromycin, targeting b. burgdorferi, without binding therapy prior to the PLS study because recent studies reported success with this protocol. It was hypothesized that: 1) b. burgdorferi had been eliminated by azithromycin; and 2) 3 weeks of atovaquone therapy was insufficient to eliminate b. microti during coinfection with b. burgdorferi. The current study was designed to investigate the efficacy of atovaquone therapy on b. microti. infection during a 3 week period and, if indicated, during an additional 6 week period.

Methods

Twenty-five patients with serologic evidence of exposure to b. microti and b. burgdorferi and chronic symptoms despite 3 weeks of atovaquone and azithromycin therapy followed by toxin-binding therapy were enrolled in an FDA and IRB approved double-blinded, placebo-controlled, crossover clinical trial. Patients were randomly assigned either atovaquone or placebo for 3 weeks in combination with CSM, and then crossed over to the other arm of the trial for 3 weeks. Treatment with atovaquone and CSM, provided on an open label basis, for 6 additional weeks followed. Symptoms and VCS scores were recorded at initiation and at the end of weeks 3, 6, 9 and 12.

Results

Twenty-one patients completed the trial, four dropped out; one for non-study related reasons, two because of CSM intolerance and one patient because of no improvement at 9 weeks. No patients showed notable symptom resolution or vision recovery at the end of week 6. After an additional 6 weeks of therapy, five patients had complete resolution of their longstanding symptoms and 16 had notable reduction in the number and/or severity of symptoms. The cohort showed a statistically significant reduction in symptom number and increase in VCS between initial and week 12 assessments. There were no adverse effects noted other than mild worsening of symptoms experienced by patients during the first few weeks of atovaquone therapy. Patients noticed clinical improvement near the end of the second 3-week course of atovaquone, and noted continued improvement during the third 3-week course of atovaquone and CSM therapy.

Discussion

The study results suggest that prolonged atovaquone therapy is required to eliminate b. microti. None of the patients had shown improvement after 3 weeks of atovaquone in combination with azithromycin therapy, after 3 weeks of binding therapy or after 3 weeks of atovaquone plus binding therapy. Six to 9 continuous weeks of azithromycin therapy are likely required for improvement in most cases. Studies of coinfected patients are needed to confirm and extend these results, particularly due to coinfection rates exceeding 10% in endemic areas. Additional issues to address include: 1) whether b. microti, like b. burgdorferi, also releases a toxin; 2) whether upregulation of anti-inflammatory cytokines triggered by b. burgdorferi toxins interfere with atovaquone's lethal mode of action on b. microti; 3) the role of pro-inflammatory cytokines in symptom induction and the course of coinfection; 4) possible extravascular sequestration of viable Babesia organisms; and 5) the role of the extrachromosomal 35 kb plastid DNA, homologous to DNA of Euglena, in maintaining viable Babesia organisms.

Sick Building Syndrome: Possible Association with Exposure to Mycotoxins from Indoor Air Fungi

HK Hudnell1, RS Shoemaker2
1US Environmental Protection Agency, Research Triangle Park, NC 27711 USA
2McCready Outpatient Services Center, Pocomoke City, MD 21851 USA

Introduction. Chronic human illness associated with residential or occupational buildings, commonly referred to as sick building syndrome (SBS), may be a multifactorial condition, involving in some cases volatile organic compounds, CO or CO2, pesticides, biologic agents, temperature and humidity, lighting, and neuropsychological status. Recent evidence indicated that the primary causative factor in a subset of SBS cases may be exposure to mycotoxins from indoor air fungi. A variety of fungal genera, including Stachybotrys, Aspergillus, Penicillium and Cladosporium, have been identified on interior cellulose materials following water intrusion. Many species have been shown to produce mycotoxins and release spores to air. Mycotoxin exposure has been associated with effects on the nervous, digestive, respiratory, cutaneous, urinary, reproductive, immune and other systems (1). Dr. Shoemaker's data from a series of cases are described to illustrate a new approach to diagnosis and treatment of mycotoxin-induced SBS. Methods. As in other biotoxin-induced chronic human illnesses for which techniques to identify toxins in tissues are unavailable, diagnosis was based on exposure potential, the presence of multiple system symptoms, and the absence of probable alternative causes of illness. Samples of fungi were observed growing in each building and analytically identified to assess exposure potential. Symptoms were systematically assessed in direct interview. A test of visual pattern detection ability, visual contrast sensitivity (VCS), was administered to each patient to assess its usefulness as an objective indicator of neurotoxicity in mycotoxicosis. Alternative explanation of illness were assessed with clinical and laboratory techniques, as well as with questions on medical history, potential for exposure to other toxins and life style. Cases were treated solely with an orally administered, non-absorbable polymer, cholestyramine (CSM), that binds salts from bile through anion exchange. CSM was previously used to successfully treat chronic illness induced by other biotoxins (2), presumably by preventing toxin recirculation through enterohepatic reabsorption, thereby enhancing toxin elimination rates. Results. One or more genera of toxin forming fungi was identified in each building. All cases reported neurologic symptoms and symptoms involving at least three other systems. All cases showed depressed VCS in the presence of normal visual acuity, indicative of a neurologic effect. No probable alternative causes of illness were identified. Following 2 weeks of CSM therapy in the absence of re-exposure, all cases showed normal VCS and at least a 90% resolution of symptoms. Relapse occurred only with re-exposure and resolved with re-treatment. Conclusions. Even in the absence of measures of airborne spore concentrations and mycotoxin levels in tissue, these results strongly support the hypothesis of mycotoxicosis in these SBS patients. Multiple system symptoms, the objective indication of a neurologic effect provided by VCS, relapse with re-exposure and successive recoveries following CSM therapy are consistent with this diagnosis. The CSM response in these chronically ill patients unresponsive to previous treatments has no known explanation other than enhancement of toxin elimination rates. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.
References. 1. HM Ammann. Is indoor mold contamination a threat to health? http://www.doh.wa.gov/ehp/oehas/mold.html
2. RS Shoemaker & HK Hudnell. Possible estuary associated syndrome: symptoms, vision and treatment. http://ehpnet1.niehs.nih.gov/docs/2001/109p539-545shoemaker/abstract.html

Differential association of HLA DR genotypes with chronic, neurotoxin-mediated illnesses: Possible genetic basis for susceptibility?

Ritchie Shoemaker, Dennis House
Center for Research on Biotoxin-Associated Illness (a not-for-profit Corporation)
500 Market Street, Suite 102, Pocomoke City, MD 24851

ABSTRACT
The recently published paradigm of chronic biotoxin-induced illness involves multiple-system symptoms, a neurologic deficit as indicated by visual contrast sensitivity measurement, and symptom resolution concomitant with vision recovery following cholestyramine treatment to bind and eliminate toxins. Chronic illness can follow exposure to toxins from Ciguatera, Pfiesteria and other dinoflagellates (dinoflagellate-related illness, DRI), Aspergillis, Penicillium, Stachybotrys and other indoor air fungi (sick building syndrome, SBS), nasal resident coagulase negative Staphylococcus (CNS), and bacteria and protozoa associated with Post-Lyme Syndrome (PLS). Because some exposed individuals acquire only acute illness or are unaffected, we suspected that susceptibility to chronic illness was conferred by particular patterns of genetic polymorphisms, perhaps those coding for antigen presentation to immune T cells. Alleles at five loci on the class II human leukocyte antigen (HLA) genome were identified with commercial PCR analyses, 2 alleles for DRB1, DQ and DRB3, and 1 allele for DRB4 and DRB5, for 200 chronically ill patients with well established exposure potentials.

In each of the four patient classes, analyses identified unique and disproportionate patterns of HLA alleles within the patients' entire sets of alleles, relative to the entire patient population and to published frequencies in a large normal population. Statistically significant differences in allele fingerprints were: DRI - DRB1-4, DQ-8, DRB4-53; SBS - DRB1-7 or 17, DQ-2, DRB3-52A; CNS - DRB1-11, DQ-7, DRB3-52B; and PLS - DRB1-15, DQ-6, DRB5-51. The specific allele pattern was present in about 90% of patients in each class, and only about 5% of patients also showed an allele pattern associated with another patient class. The few patients with illness suspected to involve two types of exposure had allele patterns associated with risk from both types of exposure. In addition, several patients with histories of illness in more than one of the patient classes showed another allele pattern, DRB1-14, DQ-5 and DRB3-52B. Preliminary data indicated that the biotoxin exposure associated with each patient class and allele pattern may also be associated with a distinct pattern of elevation in proinflammatory cytokines.

These preliminary results suggest that distinct genetic patterns may be risk factors for chronic illness from exposure to particular classes of biotoxins. Susceptibility may involve cloaked or inappropriate presentation of antigens to T cells, and ensuing ineffective antibody production. A larger patient-population study that includes a prospective component is needed to confirm the possibility that particular HLA genotypes are risk factors for acquisition of chronic, neurotoxin-mediated illness, and to investigate the possible roles of proinflammatory cytokines in the toxic modes of action. Chronic inflammation is a known risk factor for development of a variety of diseases.

Vibrotactile Threshold and Pin-Prick Sensitivity as Indicators of Subclinical Changes in Somatosensory Function: Effects of Environmental Exposure to Arsenic in Drinking Water

DA Otto1, HK Hudnell1, Y-H Li2, YJ Xia2
1US Environmental Protection Agency, Research Triangle Park, NC 27711 USA
2Institute of Endemic Disease, Huhhot, Inner Mongolia

Introduction. Peripheral neuropathy results from exposure to a variety of neurotoxic compounds. Clinical assessment of peripheral neuropathy is commonly based on electrophysio-logical measures of nerve conduction velocity (NCV). However, data on early, subclinical changes in neurologic function resulting from exposure to neurotoxicants are needed for human health risk assessment. Behavioral measures of vibrotactile thresholds (VTT) and pin-prick sensitivity (PPS) provide non-invasive methods for assessing somatosensory function that are suitable for use in field studies. VTT measures have previously identified subclinical effects from exposure to organic solvents, jet fuel, elemental mercury and organophosphate pesticides (1). The current study investigated the effects of environmental exposure to arsenic in drinking water on somatosensory function using VTT and PPS techniques. Methods. A population of farm families living in the Bamen region of Inner Mongolia chronically exposed to arsenic in well water were screened for potentially confounding factors. 309 residents (mean age 35.8 +/- 12.8 years) qualified for participation. Subjects were assigned to three exposure groups based on arsenic levels in individual wells as follows: low (<20 ug/L; N=97), medium (100-300 ug/L; N=109) and high (400-700 ug/L; N=103). VTT was measured on the dorsal surface of digit 2 and the ventral surface of digit 5 on each hand. PPS was assessed on both arms and both legs. Results. VTT was significantly elevated and PPS was significantly reduced in the high exposure group relative to both the medium and low exposure groups. The data suggested a larger exposure effect on PPS than VTT. Conclusion. These results indicate that subclinical effects of chronic arsenic exposure on somatosensory function are observable only at exposure levels well above those associated with increased risk for cancer. Differences between the VTT and PSS results suggest that small diameter unmyelinated axons with free nerve endings in superficial skin layers may be more susceptible to exposure than the larger diameter and myelinated axons with Pacinian corpuscle receptors located in deeper skin layers. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.

(1) D Mergler. Behavioral Neurophysiology: Quantitative Measures of Sensory Toxicity. In: LW Chang & W Slikker (eds). Neurotoxicology: Approaches and Methods, New York, Academic Press, 1995, ch47 pp727-736.