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Index to Abstracts from AACFS Conference October 1998


Abstracts of Papers Presented at
The Bi-Annual Research Conference of the
American Association for
Chronic Fatigue Syndrome (AACFS)

October 10-11, 1998 -- Cambridge, Massachusetts

Poster Session 5: Microbiology

Co-chairs: Dharam Ablashi DVM and Paul Levine, MD
October 10, 1998, 5:00-5:30 pm

1. Ixenobiotic toxicity associated with avital (root canal) teeth and associated with Chronic Fatigue Syndrome (CFS)
Paul R. Chenev MD.PhD, Holly Keever LMT, Elizabeth Furr MT, and Ian Hyams, MD
2. Multiplex PCR for the detection of Mycoplasma fermentans, M. hominis, and M. penetrans in patients with chronic fatigue, fibromyalgia, rheumatoid arthritis and Gulf War Syndrome
Aristo Vojdani, Al Robert Franco, and Paul Choppa
3. Absence of evidence for Borna Virus infection in Swedish patients with Chronic Fatigue Syndrome
Evengård B, Lee SW, Lind G, Lipkin I
4. Roundworm-like specimens in the sputum of Chronic Fatigue Syndrome patients and controls in open and blind analyses
Lawrence A. Klapow
5. Borna Disease Virus proteins in patients with CFS
Susan Levine MD, Ian Lipkin, MD, and Shaun Lee
6. Infective agents in patients with CFS
McGill M, Simpson K, Behan WMH, Gow JW and Behan PO
7. The relationship between Chronic Fatigue Syndrome, fibromyalgia and chemical sensitivity
Aristo Vojdani and Paul Choppa


Ixenobiotic toxicity associated with avital (root canal) teeth and associated with Chronic Fatigue Syndrome (CFS)

Authors: Paul R. Chenev MD, Ph.D.; Holly Keever LMT; Elizabeth Furr MT; and Ian Hyams, MD.

Objective:The purpose of this study is to evaluate whether or not root-canal teeth from CFS patients represent a significant source of xenobiotic toxins which have been recently associated with CFS and correlate with severity of illness. Since the pioneering work of Edward C. Rosenow, chief of bacteriology at the Mayo Clinic from 1915 - 1944, it has been known that avital or dead teeth can be a source of infection and human illness. Austin and Cook reported that 89% of avital teeth versus 4% of living teeth were infected by standard culture techniques. Advances in bacteriology, especially anaerobic culture techniques has increased that percentage to over 96% of root filled avital teeth and 98% of non-root filled avital teeth. Recent studies using electron microscopy has shown that 60% of avital tooth infections are bacterial, while 40% are fungal.

Method: Five patients with root canal teeth who met the CDC criteria for CFS were randomly chosen. An in vitro toxicity assay was performed on three extracted teeth at the University of Kentucky using previously published procedures. In brief, this procedure monitors the activity of five enzymes with and without exposure to 100ul of sterile saline in which the root canal tooth has been soaked after two previous soak solutions were discarded. These enzymes are chosen for their ability to bind ATP. The interaction of these enzymes with radioactive analogs of ATP using the technique of photo affinity labeling was measured. Toxic inhibition of the ATP binding site results in fewer radioactive byproducts of the enzymatic reaction which is detected by autoradiography of the protein reaction products. The enzymes are chosen for their sensitivity to a wide variety of toxic compounds which inhibit the interaction of the enzymes with ATP.

Result:All five root-canal teeth extracted from CFS patients exhibited significant toxicity, with an average inhibition of all five enzyme to 35% (range of 21% to 60%) of their control activity without exposure to root-canal toxins. This level of toxicity was observed with a xenobiotic toxin dose estimated at parts-per-billion present at much higher dose in the root-canal tooth. Preliminary outcome studies at one month follow-up suggest that a subset of patients respond significantly to the extraction of this xenobiotic toxicity source. Other studies, presently underway, suggest that the technique of extraction must include attention to the bony elements beneath the tooth to provide optimal outcome.

Conclusion: Recent studies have strongly suggested that xenobiotic toxicity play a substantial role in symptoms related to Chronic Fatigue Syndrome. Efforts to date have centered on the relative toxicity of the portal circulation and the inability of the liver to properly detoxify this substantial source of potential toxicity. Our study suggests that other sources of xenobiotic toxicity, such as root-canal teeth may also be the cause of xenobiotic toxicity linked to CFS. Preliminary studies underway also suggest that the technique of extraction may be critical to optimal outcome and furthermore, extractions of root-canal teeth may not always lead to clinical improvement because it is not the only source of xenobiotic toxicity.

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Multiplex PCR for the detection of Mycoplasma fermentans, M. hominis, and M. penetrans in patients with chronic fatigue, fibromyalgia, rheumatoid arthritis and Gulf War Syndrome

Authors: Aristo Vojdani, Al Robert Franco, and Paul Choppa. Immunosciences Lab., Inc. in Beverly Hills, CA and the Arthritis Center of Riverside, CA

Objective: To detect three different mycoplasma species in the blood of patients with overlapping symptomatologies.

Methods: A multiplex polymerase chain reaction (PCR) was developed to detect the presence of mycoplasma genus DNA sequences and to differentiate between three human pathogenic mycoplasma species simultaneously. One set of oligonucleotide primers which is specific for a highly conserved region among all members of the genus mycoplasma, along with three other primer sets which are specific for M fermentans, M. hominis, and M penetrans species were used in this assay. The sensitivity was determined by infecting peripheral blood mononuclear cells (PBMC) of healthy individuals with known bacterial copy numbers from each species, extracting the DNA, and subjecting 1 mg of DNA from each sample to 40 cycles of amplification. By using agarose gel electrophoresis, the detection level was determined to be 7, 7, 9, and 15 mycoplasma cells per mg of human genomic DNA for the M genus, M fermentans, M hominis, and M penetrans primer sets respectively. The assay was applied to DNA samples extracted from the PBMCs of individuals suffering from chronic fatigue syndrome (n=100), fibromyalgia (n=40), rheumatoid arthritis (n=100), Gulf War Syndrome (n=60), and control subject (n=160).

Results: The percent of positive patients with different clinical conditions but with overlapping symptomatologies for different pathogenic mycoplasmas and their comparison with control subjects is presented in the following table.

% Positive For:
 

Control
Civilians

Chronic
Fatigue

 
Fibromyalgia

Rheumatoid
Arthritis

Gulf War
Syndrome

Control
Soldiers

M. fermentans

83235 23364

M. hominis

398 1150

M. penetrans

264 730

Significant differences in the presence of these pathogenic mycoplasma species in the blood of patients with different chronic illnesses and control subjects indicate the involvement of this organism in CFS and other related disorders.

Conclusions: These results suggest that multiplex PCR provides a rapid and cost efficient procedure for screening clinical specimens for the presence of three potentially pathogenic species of mycoplasma. Detection of these mycoplasmas by highly sensitive and specific PCR suggests the role of co-pathogens for mycoplasma species in CFS, FMS, RA and GWS, and may contribute to the pathogenesis of fatigue and other symptomatologies associated with these diseases.

Choppa, PC; Vojdani, A; Tagle, C; Andrin, R; and Magtoto, L. Multiplex PCR for the detection of Mycoplasma fermentans, M hominis, and M penetrans in cell cultures and blood samples of patients with chronic fatigue syndrome. Molecular and Cellular Probes, 1998 (in press).

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Absence of evidence for Borna Virus infection in Swedish patients with Chronic Fatigue Syndrome

Evengård B, Lee SW, Lind G, Lipkin I Dept of Infectious Disease, Karolinska Institutet at Huddinge University Hospital, Stockholm, Sweden and Lab for Neurovirology, Univ of California, Irvine

Objective: A majority of patients attending a CFS-polyclinic at a clinic of infectious diseases in Stockholm report an infectious onset. Borna disease virus (BDV) is a novel neurotropic virus reported to be associated with neuropsychiatric disorders. We wanted to investigate the influence of BDV on patients diagnosed with Chronic Fatigue Syndrome.

Methods: A total of 169 patients attending the polyclinic and fulfilling CDC-criteria were investigated. Disease was judged to be severe in 22 patients, and mild in 147. Controls were healthy friends or non-blood relatives to the patients, sex- and age-matched, and random blood donors from a region 100-km northwest of Stockholm. Serum samples were collected and stored as serum and also peripheral blood mononuclear cells were isolated and frozen. ELISA and Western immunoblot were used for antibody detection. Recombinant BDV proteins N, P, and gp 18 were used in both assays. As a negative control antigen beta-galactosidase was used. A nested reverse transcriptase polymerase chain reaction (RT-PCR) was used for analysis of the presence of BDV-P nucleic acid in cells.

Results: There was a high reactivity to control protein beta-galctosidase in ELISA. No specific immunoreactivity to BDV was detected in sera or in cells.

Conclusion: We find no evidence in blood for an involvement of BDV in the pathogenesis of CFS. The high reactivity to the control antigen suggests existence of a polyclonal activation and we are still intrigued by the hypothesis of involvement of a cryptic infection.

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Roundworm-like specimens in the sputum of Chronic Fatigue Syndrome patients and controls in open and blind analyses

Author: Lawrence A. Klapow

Objectives:

  1. determine the occurrence of a suspected nematode parasite, Cryptostrongylus pulmoni in CFS patients and controls, in both open and blind trials;
  2. demonstrate skill in identification through the blind analysis; and
  3. preserve specimens for biochemical evaluation.

Methods: The distinguishing features of C. pulmoni, primarily male reproductive structures (bursa, genitals, and spicules) and female mouth parts, were first determined from specimens found in the sputum of a patient taking an anti-roundworm medication. Three-day sputum samples were obtained without the use of drugs from 28 CFS patients and 6 controls preserved in 50% ethanol). Patients were drawn from a CFS research center, general medical practices, and an Internet group. Most patients filled out a medical questionnaire. Initial microscopic examination provided candidate specimens, which were preserved in glycerin. An imaging system was used which allowed the mounted specimens to be turned over and photographed from both sides at high magnification (1200X), and various focal depths (a 12X optical video mounted to the l0X ocular of a microscope set at the 10X objective lens). A second polarizer inserted in the ocular lens, as an optical analyzer, sometimes revealed additional structures not visible with the single polarized light source technique.

Results: Specimens of C. pulmoni, in various states of decay, were found in 11 out of 28 CFS patients, but not in 6 controls, in the combined analyses. The sample size was too small for a chi-squared contingency test. In the blind analysis, 5 of the 11 CFS patients, but none of the 6 controls, were positive. The probability of getting as good a result in a coin toss (random guess) model was 0.01. Of the 11 positives, 10 contained 1 specimen and the other had 2. Re-testing of 3 initially negative patients resulted in 1 positive. Specimens were small: 200 to 500 microns long. Identification required careful evaluation of the photographic images. Three positive patients reported borderline to low-grade eosinophilia (6-9%). Stool tests were negative.

Conclusion: Decayed specimens of a suspected roundworm, Cryptostrongylus pulmoni (provisional) "the hidden lung worm", were found in the naturally expelled sputum of 39% of CFS patients, but not in 6 controls. Low test sensitivity suggests that actual occurrence is likely to be higher. A larger test design will be needed to evaluate the possible association of C. pulmoni with CFS. A random guess model suggested that skill in identifying C. pulmoni, rather than chance, governed the results of the blind analysis. Current diagnostic methods would not be expected to detect C. pulmoni. The usual indicators of nematode infections, high-grade eosinophilia and positive stool tests, were not apparent. In addition to being decayed, specimens were also very small and rare, and required specialized imaging techniques for identification. Roundworm infections can produce immune abnormalities like those reported in CFS, including low serum cortisol, altered anti-viral responses, eosinophil anomalies, and altered cholinergic processes.

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Borna Disease Virus proteins in patients with CFS

Authors: Susan Levine, MD; Ian Lipkin, MD; and Shaun Lee

Objective: Bornavirus is a member of a newly recognized virus family, Bornaviridae, and is neurotropic for a wide range of animal species, including birds, rodents, horses and humans. Although little is known about its mode of transmission and it has not been clearly linked to any human disease, an association between bornavirus and neuropsychiatric diseases has been suggested. We sought to study the prevalence of Borna Disease Virus (BDV) proteins in a non-select group of CFS patients.

Methods: 110 serum samples from 77 CFS patients and 33 healthy controls were examined for evidence of BDV infection using an ELISA based on 3 recombinant BDV proteins, nucleoprotein (N), phosphoprotein (P), and matrix protein (M). An irrelevant protein, beta-galactosidase (Beta-gal), was used as a control for the specificity of immunoreactivity to BDV proteins. Initial screening for reactivity was performed at a dilution of 1:25.

Results: 15 samples were immunoreactive to one or more recombinant BDV proteins (#3, #6, #8, #11, #17, #18, #23, #24, #27, #33, #36, #41, #59, #93, #108) and were re-assayed in a secondary screen using the same parameters. Of these, samples #3 and #33 were normal controls. The six samples which were immunoreactive in the secondary screen to 2 or more BDV proteins were selected for a tertiary screen using serum dilutions of 1:25 and 1:50. All samples selected for rescreening had consistent reactivity in consecutive assays, and Optical Density measurements were consistent in all 3 screenings. Dilution of serum from 1:25 to 1:50 in the tertiary assays resulted in a measurable decrease in O.D. measurements in the immunoreactive samples. All samples were immunoreactive to beta-galactosidase, and may reflect differences in methods used in serum collection. Sample #93 appeared to be the most immunoreactive, and we believe it would be worthwhile to further study serum from this patient using Western Blot and Immuoprecipitation, as well as mononuclear cells using molecular methods of detection of viral nucleic acids such as PCR methods.

Conclusion: Although no serum sample demonstrated specific immunoreactivity to BDV proteins (all were immunoreactive to beta-gal) several samples were immunoreactive to 2 or more BDV proteins. Immunoreactivity to 2 or more BDV proteins is consistent with BDV infection in other systems. If clinical correlation supports a potential role for viral infection we recommend pursuing further studies.

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Infective agents in patients with CFS

Authors: McGill M, Simpson K, Behan WMH, Gow JW and Behan PO.
Glasgow University Dept of Neurology, Southern General Hospital, Govan Rd, Glasgow G51 4TF, Scotland.

Objective: Chronic fatigue syndrome (CFS) is often thought to follow a viral-like episode. Although many infectious agents have been associated with CFS, no single agent has been isolated or identified. As part of our long-term programme to elucidate the pathogenesis of CFS, in this study six specific infectious agents were sought in tissue from patients with CFS. These include the DNA viruses varicella zoster virus and hepatitis B; the RNA viruses Borna disease virus, enteroviruses and Influenza B; and Brucella, as chronic Brucellosis may present with a condition similar to CFS. The study was carried out in order to confirm or eliminate these organisms as possible chronic pathological agents, to identify patients with recurrent or chronic infection which could indicate any immune system abnormality and to identify subgroups of patients.

Methods: A two-year double-blind study was undertaken with 64 patients with CFS, and 34 carefully age/sex/geographical location/time-of-sampling matched controls. Following a full clinical screening, peripheral blood samples were taken for peripheral blood mononuclear cell (PBMC) isolation and, in certain cases, lumbar puncture for cerebral spinal fluid or liver biopsy was performed. PBMC nucleic acids were prepared from patients and controls prior to PCR amplification for Brucella and the DNA viruses, and RT-PCR amplification for the RNA viruses, followed by Southern blot analyses and hybridisation with internal oligonucleotide probes.

Results: The data presented here represents PCR analyses on nucleic acids extracted from PBMC samples.

 

CFS Patient

Control

VCV:

3 +ve

0 +ve

hepatitis B:

4 +ve

0 +ve

BDV:

3 +ve

0 +ve

enteroviruses:

0 +ve

0 +ve

influenza B:

0 +ve

0 +ve

Brucella:

2 +ve

0 +ve

Conclusion: Of the 64 patients studied, 3% had a current Brucella infection, 6% were infected with hepatitis B, 4.6% with VZV and 4.6% with BDV. Overall, 18% of the patients had evidence of infection, although that figure would be reduced to 14% if the VZV positive cases were due to a sub-clinical reactivation of latent virus rather than a recent infection. None of the infectious agents were present in a significantly high number of cases of CFS and none of the patients had evidence of dual infection. The data highlights one difficulty of working with patients with CFS -- patients who have been diagnosed as having the disorder may be suffering from undiagnosed less common infections. Data from subsequent research into CFS may be inaccurate due to a dilution of the true patient cohort.

This study was funded by the ME Association and the Linbury Trust.

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The relationship between Chronic Fatigue Syndrome, fibromyalgia and chemical sensitivity

Authors:Aristo Vojdani and Paul Choppa. Immunosciences Lab., Inc. Beverly Hills, CA.

Objective: Overlapping symptomatologies between Chronic Fatigue Syndrome and Chemical Sensitivity have been observed by different investigators. Therefore, it is of great importance to develop biomarker(s) for possible differentiation between viral induced CFS (without sensitivity to chemicals) versus chemically induced CFS. Since interferon induced proteins 2-5A Synthetase and protein kinase RNA (PKR) have been implicated in viral induction of CFS, the objective of this study was to utilize 2-5A and PKR activity for the differentiation between CFS induced by either viruses or chemicals.

Methods: Based on the CDC definition and criteria, twenty CFS patients who were positive for viral genome(s) [mainly HHV6; HTLVl, EBV, and CMV] and did not have any history of exposure to chemicals were included in this study. For comparison, the second group of patients consisted of twenty individuals from the same geographical area who were negative for viral genomes but had been exposed to methyl tertiary-butyl ether concentration of up to 70 ppm and benzene concentration up to 14 ppm. All patients complained of fatigue and other symptoms which were overlapping with the viral genome positive CFS group. From all 40 patients, blood was drawn, leukocyte extract was prepared and assayed for 2-SA Synthetase and PKR activity.

Results: Clinical specimens which were positive for viral genomes showed from 2.2-38.7 and 1.3-13.5 fold increase in 2-5A and PKR activities over the background of the healthy controls respectively. Similarly the second group (negative for viral genomes, but exposed to chemicals) showed 1.1-29.2 fold increase for 2-5A Synthetase and 1.3-11.6 fold increase for PKR when they were compared to healthy subjects. To elucidate mechanisms involved in viral versus chemical induction of 2-5A Synthetase and PKR, MDBK cell lines were cultured in the presence or absence of HHV6, MTBE, or Benzene. In addition, MDBK cells were incubated with heat shock proteins and interferon-beta. 2-5A and PKR activities were measured in all the above conditions. A clear induction of 2-5A and PKR was observed when MDBK cells were exposed to HHV6, MTBE, and Benzene. This induction was more significant with HSP90, HSP70, and INF-alpha, indicating their involvement in the mechanism of action.

However, when MDBK cells were incubated either with MTBE + Benzene or HHV6 in the presence or absence of anti INF-beta or anti HSP-70, the activities of both 2-5A and PKR in HHV6 infected MDBK cells were inhibited by more than 90% by anti INF-beta, and only 20% by addition of anti HSP70. While in MTBE + Benzene exposed cells anti INF-beta reduced the activity of these enzymes by 40% and anti HSP7O by more than 90%. This indicates an involvement of INF-beta in viral induction 2-5A and PKR and HSP involvement in chemical induction of these enzymes.

Conclusion: We conclude that 2-5A and PKR are not only biomarkers for viral induction of CFS, but biomarkers for other stressors inducing CFS which include MTBE and Benzene. Interferon-beta is involved in the induction of 2-5A and PKR in CFS patients, and HSP is more involved in this mechanism of action induced by toxic chemicals.

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