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Townsend Letter 2008 April Issue, vol. 295: 74-84.
Systemic Intracellular Bacterial Infections (Mycoplasma,
Chlamydia, Borrelia species) in Neurodegenerative
(MS, ALS, Alzheimer’s) and Behavioral (Autistic Spectrum
Disorders) Diseases
Prof. Garth L. Nicolson
The Institute for Molecular Medicine
Dept. of Molecular Pathology, P.O. Box 9355
S. Laguna Beach, California 92652
Phone: +1 949-715-5978 Fax: +1 714 596-3791;
Email: gnicolson@immed.org Website: www.immed.org
Abstract
Patients with neurodegenerative diseases and behavioral disorders often have systemic bacterial, viral and/or fungal
infections that are important in disease progression and severity. We and others have examined patients with various neu-
rodegenerative and behavioral neurological conditions, such as Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis
(MS), Alzheimers Disease (AD) and Autistic Spectrum Disorders (ASD, including Autism, Attention Deficit Disorder,
Asperger Syndrome), and found evidence for systemic intracellular bacterial and viral infections in a majority of patients.
For example, examination of blood for evidence of Mycoplasma species, Chlamydia pneumoniae, Brucella species, Bor-
relia burgdorferi and other infections by serology, Western blot or polymerase chain reaction revealed high incidences of
systemic co-infections that were not found in control subjects (P<0.001). The results were compared to other chronic ill-
nesses where neurological manifestations are often found, such as Chronic Fatigue Syndrome/Myalgic Encephlomyopathy
(CFS/ME), Fibromyalgia Syndrome (FMS), Lyme Disease and Gulf War Illnesses. Most of these chronic illness patients
also had multiple intracellular bacterial infections compared to control subjects (P<0.001), and the most common co-
infection found was Mycoplasma species in all of the conditions examined. In contrast, in the few control subjects that
tested positive, only single infections were found. The results suggest multiple chronic intracellular bacterial (and viral)
infections are common features of neurodegenerative and behavioral disorders, and treatment regimens should address the
multiple infections present in these conditions.
1. Introduction
Neurodegenerative diseases are chronic degenerative diseases of the Central Nervous System (CNS) that often cause de-
mentia in the aging population.! For the most part the causes and mechanisms of this collection of brain diseases remain
largely unknown, and they are increasing in incidence in the developed as well as the underdeveloped world.1!These dis-
eases are characterized by molecular changes in nerve cells that result in nerve cell degeneration and ultimately nerve dys-
function and cell death, resulting in neurological signs and symptoms and ultimately dementia.1,2!
There appears to be a genetic link to neurodegenerative diseases, but the genetic changes that occur and the
changes in gene expression that are found in these diseases are complex and not related to simple genetic mutations, dele-
tions or amplifications.1! In addition to genetic changes and changes in gene expression, it is thought that nutritional defi-
ciencies, head trauma, environmental toxins, chronic bacterial and viral infections, autoimmune immunological responses,
vascular diseases, accumulation of fluid in the brain, changes in neurotransmitter concentrations and other causes are in-
volved in various neurodegenerative diseases.1-5 One of the biochemical changes found in essentially all neurological de-
generative diseases is the over-expression of oxidative free radical compounds (oxidative stress) that cause lipid, protein
and genetic structural changes.3,4
An attractive model for neurodegeneration resulting neurological disease involves the action of toxic products
produced as a result of chronic bacterial and/or viral infections.6,7 Infectious agents may enter the CNS within infected
migratory macrophages, or they may gain access by transcytosis across the blood-brain-barrier or by intraneuronal trans-
fer from peripheral nerves.6 Cell wall-deficient bacteria, principally species of Mycoplasma, Chlamydia, Coxiella, Bru-
cella, Borrelia, among others, are candidate infectious agents that may play an important role in neurodegenerative dis-
eases.8 Such infections may also cause disease progression, and since they are usually systemic, they could affect the im-
mune system and other organ systems, resulting in systemic signs and symptoms.9
2. Methods
Blood Collection
Blood was collected, immediately brought to ice bath temperature and shipped with wet ice by air courier to the
Institute for Molecular Medicine for analysis. All blood samples were blinded. Whole blood was used for preparation of
sera or DNA using Chelex as previously described.10,11 Multiple tests were performed on all patients and control subjects.
Western Blot of Borrelia burgdorferi
Patients were recruited who were previously tested for Borrelia burgdorferi using Western Blot analysis.12,13
Laboratory results were examined, and criteria for a positive Western blot was that at least two of the Borrelia burgdorferi
genus-specific antigens (18K, 23K, 30K, 31K, 34K, 37K, 39K, 83K and 93K) were reactive in Western blots.
Amplification of Gene Sequences by PCR
Amplification of the target gene sequences by Polymerase Chain Reaction (PCR) was accomplished as previously
described.10,11 Negative and positive controls were present in each experimental run. The amplified samples were sepa-
rated by agarose gel electrophoresis. After denaturing and neutralization, Southern blotting was performed to confirm the
PCR product. 10,11 Multiple PCR primer sets were used for each species tested to minimize the chance that cross-reacting
microorganisms were detected.
Statistics
Subjects' demographic characteristics were assessed using descriptive statistics and students' t-tests (independent
samples test, t-test for equality of means, 2-tailed). Pearson Chi-Square test was performed to compare prevalence data
between patients and control subjects.
3. Amyotrophic Lateral Sclerosis (ALS)
ALS is an adult-onset, idopathic, progressive degenerative disease affecting both central and peripheral motor neurons.
Patients with ALS show gradual progressive weakness and paralysis of muscles due to destruction of upper motor neurons
in the motor cortex and lower motor neurons in the brain stem and spinal cord, ultimately resulting in death, usually by
respiratory failure.14,15 The overall clinical picture of ALS can vary, depending on the location and progression of patho-
logical changes found in nervous tissue.16
In ALS patients the role of chronic infections has attracted attention with the finding of enterovirus sequences in a
majority of spinal cord samples by PCR.17,18 Although others have failed to detect enterovirus sequences in spinal cord
samples from patients with or without ALS,19 infectious agent(s) may play a role in the etiology of ALS. We studied the
presence of systemic microbial infections in a preliminary number of ALS patients.20 We found that 8/8 Gulf War veter-
ans diagnosed with ALS from three nations had systemic mycoplasmal infections. All but one patient had M. fermentans
infections, and one veteran from Australia had a systemic M. genitalium infection. In 22/28 nonmilitary ALS patients
from the USA, Canada and Great Britain we also found blood mycoplasmal infections. Of the mycoplasma-positive ci-
vilian patients who were further tested for M. penetrans, M. fermentans, M. hominis and M. pneumoniae, most were posi-
tive for M. fermentans (13/22, 59%), but we did find other Mycoplasma species, such as M. hominis (7/22, 31%) and M.
pneumoniae infections (2/22, 9%). Two civilian ALS patients had multiple mycoplasmal infections (M. fermentans plus
M. hominis, 9%). Multiple mycoplasmal infections were not found in the military patients with ALS. The difference in
incidence of mycoplasmal infections between ALS patients and control subjects was highly significant (P<0.001).20 The
incidence of various chronic bacterial and viral infections in ALS is shown in Fig. 1. The other type of infection that is
commonly found in ALS is Lyme Borrelia burgdorferi (Fig. 1). Thus a byproduct of Lyme Disease may be progression
to ALS, but this is probably only possible in some Lyme Disease patients who have the genetic susceptibility genes for the
neurodegenerative disease.21
0%
20
40
60
80%
Percentage of ALS pts with Chronic Infections
Mycoplasma
all species
<15%
HHV-6
Virus
<15%
Chlamydia
pneumoniae
Borrelia
burgdorferi
Echo-7
Enterovirus
Hepatitis
Virus
~70%
Prof. G. L. Nicolson
Institute for Molecular Medicine
<25%
<5%
>85%
Figure 1. Percent incidence of systemic bacterial and viral infections in 46 patients with Amyotrophic Lateral Sclerosis (ALS). The
results were determined by Western blot or PCR.
ALS patients also have other chronic infections, including Human Herpes Virus-6 (HHV-6), Chlamydia pneumoniae and
as mentioned above, Borrelia burgdorferi but rarely hepatitis virus (Fig. 1). Similar to the possible role of enteroviruses in
the pathogenesis of ALS, the exact role that the other infections play in the pathogenesis or progression of ALS is not
known. They could be cofactors in the pathogenesis of ALS, or they could simply be opportunistic infections that cause
morbidity in ALS patients, such as the respiratory, rheumatic symptoms and other problems often found in ALS patients.
They could also be involved in the progression of ALS rather than in its inception.
Although the exact cause of ALS remains unknown, there are several hypotheses on its pathogenesis: (a) accu-
mulation of glutamate causing excitotoxicity; (b) autoimmune reactions against motor neurons; (c) deficiency of nerve
growth factor; (d) dysfunction of superoxide dismutase due to mutations; and (e) chronic infection(s).16-23 Future studies
should determine more precisely the role of chronic bacterial and viral infections in ALS pathogenesis and progression.
4. Multiple Sclerosis (MS)
Multiple Sclerosis is a disease of the nerves of the central nervous system, and it can occur in young as well as older peo-
ple. The nerves in various parts of the brain are covered by a protective insulation containing the protein myelin and other
proteins imbedded in a lipid sheath so that the electrical impulses that cause nerve conduction are protected. In MS, in-
flammation and the presence of autoimmune antibodies against myelin and other nerve cell antigens causes the protective
sheath to break down (demyelination), resulting in decrease or loss of electrical impulses along the nerve.
In progressive MS the nerve cells are damaged additionally by the deposition of plaques on the nerve cells to the
point where nerve cell death occurs. There is also breakdown of the blood-brain barrier associated with local inflamma-
tion caused by glial cells.24,25 The clinical results of demyelination and blood-brain barrier lesions are variable but usually
include impaired vision, alterations in motor, sensory and coordination systems and cognitive dysfunction. Often these are
cyclic (relapsing-remitting) over some time, but a subgroup of patients’ progress more rapidly.25
A possible infectious cause for MS has been under investigation for approximately the last decade.25-27 Epidemi-
ological and twin studies suggest that MS is acquired not inherited. Since more than 90% of MS patients show immu-
nological and cytokine characteristic of infection, MS patients have been examined for various viral and bacterial infec-
tions. One of the most common findings is the presence of Chlamydia pneumoniae in MS brains,28-30 although this has not
been found in every study.31,32 A possible reason for this is that other infections could also be involved. In addition to
Chlamydia pneumoniae found in some studies, MS patients could have Mycoplasma species, Borrelia burgdorferi,
Chlamydia pneumoniae and other infections.
Recent research at the Institute for Molecular Medicine and elsewhere has shown that autoimmune responses to
nerve cell proteins may be caused, in part, by intracellular bacterial infections. As many as 80% of MS patients may have
intracellular bacterial infections caused by Mycoplasma, Chlamydia and other cell wall-deficient bacteria species that
were found only at low incidence in age-matched subjects (P<0.001). Additional bacterial infections, such as Borrelia
burgdorfii (Lyme Disease), and other intracellular bacterial infections may also be involved in some MS cases (Fig. 2).
When these infections are released from cells, they contain host cell antigens in their exterior membranes, and these nor-
mal cell membrane antigens could stimulate autoimmune responses. Alternatively, the microorganisms may express anti-
gens that mimic normal surface antigens.
Viruses may also be involved in MS.33 Certain viruses have been found at high incidence in MS patients, such as
human herpes virus-6 (HHV-6).33 We have also found this virus in the systemic circulation of MS patients (Fig. 2), sug-
gesting that it might be involved in the pathogenesis of MS. Viruses may stimulate autoimmune responses when they kill
cells, resulting in release of normal antigens into the surrounding extracellular environment.
0%
20
40
60
80%
Percentage of MS pts with Chronic Infections
Mycoplasma
all species
~30%
HHV-6
Virus
<15%
Chlamydia
pneumoniae
Borrelia
burgdorferi
Echo-7
Enterovirus
Hepatitis
Virus
0%
Prof. G. L. Nicolson
Institute for Molecular Medicine
~35%
<10%
>50%
Figure 2. Percent incidence of systemic bacterial and viral infections in 65 patients with Multiple Sclerosis (MS). The results were
determined by Western blot or PCR.
Since infections usually stimulate immunological responses, the presence of intracellular bacterial infections in
nerve cells, in particular, may stimulate autoimmune responses against nerve cell antigens. In the case of MS some 20 dif-
ferent bacterial and viral infections have been found, but the link between these infections and the pathogenesis of MS is
still being debated.34 Perhaps this is the reason that one or even a few types of infections cannot be linked to every case of
MS. That, however, does not prove that infections, in general, are not linked to the pathogenesis of MS.
Does other evidence suggest that infections may be involved in the pathogenesis of MS and other neurological
diseases? The answer to this question is most certainly, yes. These diseases can progress to a fatal phase, especially when
intracellular infections are found.29 Upon autopsy intracellular bacteria, such as C. pneumoniae and Mycoplasma species,
have been found at high levels inside nerve cells in the CNS,34,35 The presence of such bacteria has been linked to various
neurological diseases.29,30 In addition, control infection of non-human primates with cell-invading bacteria, such as Myco-
plasma fermentans, results in a fatal disease with neurological complications.36 When these infected brains are examined
at autopsy, the Mycoplasma fermentans can be found in brain tissue. 36
5. Alzheimer’s Disease (AD)
Alzheimer’s Disease (AD), the most common cause of dementia, is a collection of brain disorders usually found in older
people. The disease is characterized by slow, progressive loss of brain function, especially notable by lapses in memory,
disorientation, confusion, mood swings, changes in personality, language problems, such as difficulty in finding the right
words for everyday objects, loss of behavioral inhibitions, loss of motivation, and paranoia. The prognosis and course of
AD varies widely, and the duration of illness can be a few years to over 20 years in duration. During this time the parts of
the brain that control memory and thinking are the first affected, followed by other brain changes that ultimately result in
brain cell death.37 AD is characterized by distinct neuropathological changes in the brain. Among the most notable are
the appearance of plaques and tangles of neurofibrils within brain nerves that affect nerve synapses and nerve-nerve cell
communication. Both of these structures involve the deposition of altered amyloid proteins, called Ab proteins.38,39
Although the cause of AD is not known to any certainty, the formation of the amyloid plaques and neurofiber tan-
gles may be due to genetic defects and resulting changes in the structure of Ab proteins, neurotoxicity caused by chemi-
cals or other toxic events, imflammatory responses, oxidative stress and increases in ROS, loss of nerve trophic factors
that are important in nerve physiology and loss of nerve cell transmission.38-42
Brain infections in AD have only recently become an important topic.43,44 One pathogen that has attracted consid-
erable attention is Chlamydia pneumoniae.45,46 This intracellular bacteria has a tropism for neural tissue,46 and it has been
found at high incidence in the brains of AD patients by PCR and immunohistochemistry methods. C. pneumoniae bacte-
ria have been found in nerve cells in close proximity to neurofibrillary tangles.47,48 The infection results in endothelial cell
invasion and promotes the transmigration of monocytes through human brain endothelial cells into the brain paren-
chyma.49 Although C. pneumoniae has been found in the brains of most AD patients studied,42,46 and this infection results
in amyloid beta (Abeta) plaque formation in mice injected with C. pneumoniae,50 some studies have not found an associa-
tion with Alzheimer’s using PCR51 or immunohistochemistry.52
In addition to C. pneumoniae, evidence has been forthcoming that Alzheimer’s Disease patients also have other
infections, such as Lyme Disease Borrelia burgdorferi.53 This infection has been confirmed in Alzheimer’s Disease by
serology, culture, Western blot and immunofluorenscence.54-56 In fact, the presence of intracellular infections like Borre-
lia burgdorferi found in AD are thought by MacDonald57 to be the primary event in the formation of AD amyloid plaques
by forming “congophilic cores” that attract amyloid materials. In addition, the induction of ROS, lipid peroxidation and
the breakdown of the lysosomal membrane releasing lysosomal hydrolases are also thought to be important in amyloid
deposition.58 Almost most reports show that AD nerve cells are positive for Borrelia burgdorferi in AD,53-57 but there are
also some negative reports.59 As expected, Borrelia burgdorferi coinfections are found in AD, and an interesting relation-
ship has developed between the presence of Borrelia burgdorferi and Herpes Simplex Virus-1 (HSV1) in AD.60 It had
been noted previously that HSV1 but not a related neurotrophic virus (Varicella Zoster Virus) was found often in AD
brains and may be linked to patients who have the AD risk factor apoE4 allele.61,62 HSV1 is thought to be involved in the
abnormal aggregation of beta amyloid or Abeta within the brain by reducing the amount of full length amyloid precurser
protein and increasing the amount of the Abeta fragment from this precursor.63
6. Autistic Spectrum Disorders (ASD)
Children with Autistic Spectrum Disorders, such as Autism, Attention Deficit Disorder, Asperger Syndrome, etc., gener-
ally suffer from an inability to properly communicate, form relationships with others and respond appropriately to their
environment. Such patients do not all share the same signs and symptoms but tend to share certain social, communica-
tion, motor and sensory problems that affect their behavior in predictable ways. These children often display repetitive
actions and develop troublesome fixations with specific objects, and they are often painfully sensitive to certain sounds,
tastes and smells.64,65 The signs and symptoms of ASD are thought to be due to abnormalities in brain function or struc-
ture. In some ASD patients there are also a number of other less specific chronic signs and symptoms. Among these are
fatigue, headaches, gastrointestinal and vision problems and occasional intermittent low-grade fevers and other signs and
symptoms that are generally excluded in the diagnosis of ASD.
The causes of ASD are unknown and may include genetic defects, heavy metal, chemical and biological expo-
sures, among others, and are probably different in each patient.64,65 However, among ASD patients there may be similari-
ties in genetic defects and environmental exposures that are important in patient morbidity (sickness) or in illness progres-
sion. Other chronic illnesses have some of the same chronic signs and symptoms, suggesting that there may be some
overlap in the underlying causes of these conditions or at least in the factors that cause illness or morbidity or illness pro-
gression.
Chronic infections appear to be an important element in the development of ASD.66 Such infections are usually
held in check by immune surveillance, but they can take hold and become a problem if they can avoid host immunity and
penetrate and hide in various tissues and organs, including cells of the CNS and peripheral nervous system. When such
infections occur, they may cause many of the complex signs and symptoms seen in various chronic illnesses.66-68 Changes
in environmental responses and increased titers to various endogenous viruses as well as bacterial and fungal infections
have been commonly seen in ASD along with the presence of heavy metals.64,65
In ASD there is an interesting but widely contested relationship between the disease, heavy metals and vaccines.
ASD patients often show their first signs and symptoms after multiple childhood immunizations.64 Rimland64 noted that
the sharp rise in Autism rates only occurred after the multiple vaccine MMR came into widespread use. In the U.S. chil-
dren typically receive as many as 33 vaccines before they can enroll in school, a dramatic increase in the use of childhood
vaccines over the last few decades. Such vaccines often contain mercury and other preservatives.65 Commercial vaccines
have been examined for contaminating microorganisms, and one study found that approximately 6% of commercial vac-
cines were contaminated with Mycoplasmas.69 Thus we examined the extent of intracellular bacterial infections in patients
with ASD. We were aided in this examination by data that we collected on families of Gulf War veterans where there was
a documented, deployment-associated Mycoplasma fermentans infection and a high incidence of Autism in their children
after the infected veteran returned to the home.70
Previously we found that veterans of the Gulf War with chronic fatiguing illness (GWI) exhibited multiple non-
specific signs and symptoms.71,72 Upon examination, the signs and symptoms of GWI were indistinguishable from civilian
patients diagnosed with Chronic Fatigue Syndrome/Myalgic Encephalomyopathy (CFS/ME),71,72 except for symptomatic
children aged 3-12 who were also diagnosed with Autism or Attention Deficit Hyperactivity Disorder (ADHD), two dis-
orders that fall under ASD.73 In our study 45 of 110 GWI patients or ~42% had mycoplasmal infections (Fig. 3), and al-
most all of these (37 out of 45 or ~82%) were single infections (one species of Mycoplasma).73 M. fermentans was found
in ~85% of these single infection cases (Fig. 3). When the few multiple infection cases were examined, most were found
to have combinations of M. fermentans plus either M. pneumoniae, M. hominis or M. genitalium. In contrast, in healthy
control subjects only 6 of 70 subjects (8.5%) were positive for any mycoplasmal infection, and all of these were single
infections of various types.70,73 Comparing GWI patients and non-symptomatic control subjects, there was a significant
difference in the incidence of mycoplasmal infections (P<0.001). However, differences in infection incidence or species
of mycoplasmal infection between male and female GWI patients or male and female control subjects were not seen. 70,73,74
In family members of Gulf War veterans with GWI there was evidence of illness and Mycoplasma transmission.
We found that 57/107 (53.2%) of these family members from families with one or more Gulf War veteran diagnosed with
GWI and with a positive test for a mycoplasmal infection showed symptoms of CFS/ME. Among the CFS-symptomatic
family members, most (40/57 or 70.2%) had mycoplasmal infections compared to the few non-symptomatic family mem-
bers who had similar infections (6/50 or 12%) (Fig. 3). When the incidence of mycoplasmal infection was compared
within families, the CFS/ME family members were more likely to have mycoplasmal infections compared to non-
symptomatic family members (P<0.001).70 Symptomatic children (mostly diagnosed with Autism and ADD) were also
infected with the same specie of Mycoplasma at high incidence (usually M. fermentans), and this was not seen in aged-
matched control subjects. Although some non-symptomatic family members did have mycoplasmal infections (5/50 or
10%), this was not significantly different from the incidence of mycoplasmal infections in healthy control subjects (6/70
or 8.5%) (Fig. 3).70
0%
20%
40%
Percentage of Patients/Subjects with Mycoplasmal Infections
myco+CFS/
ME
myco+Gulf
War Illness
myco+GWI
CFS-Family Members
myco+GWI-Healthy
Family Members
myco+Healthly
Controls
n=100
n=50
80%
60%
n=550
n=110
n=60
myco+GWI
CFS-Family Children
n=35
Figure 3. Percent incidence of mycoplasmal infections in family members of veterans with Gulf War Illnesses. The results were
determined by PCR.70
The Mycoplasma species was also similar between GWI patients and their CFS/ME-symptomatic family mem-
bers. In 45 mycoplasma-positive CFS/ME-symptomatic family members, most (31 out of 40 or 77.5%) had single species
infections (almost all M. fermentans), similar to the mycoplasma-positive Gulf War veterans (37 out of 45 or 82%).
These results were highly significant (P<0.001). We did not find differences in the incidence of infection or type of in-
fections between males and females, children versus adults or spouses versus other family members.70 However, similar
to previous reports, the time of onset of CFS/ME illness after the Gulf War tended to be shorter in spouses than other
family members, but these differences did not achieve significance.
We next examined a small cohort of ASD patients in Central California.73 This comprised 28 patients aged 3-12
who were diagnosed with ASD. Many of these children had at least one parent with a chronic illness, and the most com-
mon diagnosis of adults or adolescents in the same family was CFS/ME or Fibromyalgia Syndrome. When the ASD pa-
tients were examined for mycoplasmal infections, 15 children tested positive (54%) for mycoplasmal infections. However,
in contrast to the children of GWI patients who for the most part had only one type of mycoplasmal infection, M. fermen-
tans, the Central California group that tested positive for a variety of Mycoplasma species. We also tested a few siblings
without apparent signs and symptoms, and for the most part few had these infections (5/41 subjects or 12%).73 Similar
results were found in the Gulf War veterans' families where 12% of non-symptomatic family members had mycoplasmal
infections.70
0%
20
40
60
80%
Percentage of ASD pts with Chronic Infections
Mycoplasma
all species
<30%
HHV-6
Virus
<10%
Chlamydia
pneumoniae
Borrelia
burgdorferi
Staph
aurius
Hepatitis
Virus
<2%
Prof. G. L. Nicolson
Institute for Molecular Medicine
20-30%
<2%
50-60%
Figure 4. Percent incidence of bacterial and viral infections in 48 patients with Autistic Spectrum Disorders (ASD).75 The range indi-
cates results from different laboratories. Incidence was determined by Western blot, serology or PCR.
In another study we examined the blood of 48 ASD patients from Central and Southern California and found that
a large subset (28/48 or 58.3%) of patients showed evidence of Mycoplasma spp. infections compared to two of 45 (4.7%)
age-matched control subjects (Odds Ratio=13.8, P<0.001).75 Since ASD patients had a high prevalence of one or more
Mycoplasma species and some also showed evidence of infections with Chlamydia pneumoniae, we examined ASD pa-
tients for other infections (Fig. 4). In addition, the presence of one or more systemic infections may have predisposed
ASD patients to other infections, thus we examined the prevalence of C. pneumoniae (4/48 or 8.3% positive, Odds Ra-
tio=5.6, P<0.01) and Human Herpes Virus-6 (HHV-6, 14/48 or 29.2%, Odds Ratio=4.5, P<0.01) co-infections in ASD
patients. We found that Mycoplasma-positive and –negative ASD patients had similar percentages of C. pneumoniae and
HHV-6 infections, suggesting that such infections occur independently in ASD patients. Control subjects also had low
rates of C. pneumoniae (1/48 or 2.1%) and HHV-6 (4/48 or 8.3%) infections, and there were no multiple infections in
control subjects. The results indicated that a large subset of ASD patients show evidence of bacterial and/or viral infec-
tions (Odds Ratio=16.5, P<0.001).75
7. Chronic Fatigue Syndrome (CFS/ME)
Chronic fatigue is reported by 20% of all patients seeking medical care.76 It is associated with many well-known medical
conditions and may be an important secondary condition in several chronic illnesses. Although chronic fatigue is associ-
ated with many illnesses, CFS/ME and Fibromyalgia Syndrome (FMS) are distinguishable as separate syndromes based
on established clinical criteria.77 However, their clinical signs and symptoms strongly overlap. CFS/ME is characterized
by unexplained, persistent long-term disabling fatigue plus additional signs and symptoms, whereas patients with FMS
additionally suffer from muscle pain, tenderness and soreness.78 In patients with either diagnosis other conditions that can
explain their signs and symptoms are absent; thus in many patients with overlapping signs and symptoms it is difficult to
make a clear distinction between a diagnosis of CFS/ME and FMS.
Most CFS/ME and FMS patients have immunological abnormalities and infections.67,68 Thus CFS/ME patients
can be subdivided into clinically relevant subcategories that may represent different disease states or co-morbid conditions
or illnesses.79 An important subset of CFS/ME patients is characterized by the presence of chronic bacterial and viral in-
fections.10,11,66-68 Identifying systemic infections in CFS/ME patients, such as those produced by Mycoplasma species,
Chlamydia pneumoniae, Brucella species, Borrelia burgdorferi and HHV-6 infections (Fig. 5), is likely to be important in
determining the treatment strategies for these CFS/ME patients.11,79-81
0%
20
40
60
80%
Percentage of CFS pts with Various Bacterial/Viral Infections
Mycoplasma
all species
10-30%
HHV-6
Virus
8-15%
Chlamydia
pneumoniae
Borrelia
burgdorferi
Echo-7
Enterovirus
Brucella
spp.
0-2%
Prof. G. L. Nicolson
Institute for Molecular Medicine
10-20+%
<10-15%
50-60%
Figure 5. The incidence of various bacterial and vial co-infections in 100 patients with CFS/ME. The bars indicate the range of val-
ues found in different independent studies. Incidence determined by Western blot or PCR tests of blood.
Using the blood of 100 CFS/ME patients and forensic polymerase chain reaction we found that a majority of pa-
tients show evidence of multiple, systemic bacterial and viral infections (Odds Ratio = 18.0, 95% CL 8.5-37.9, P< 0.001)
that could play an important role in CFS/ME morbidity.11,79 CFS/ME patients had a high prevalence of one of four Myco-
plasma species (Odds Ratio = 13.8, 95% CL 5.8-32.9, P<0.001) and often showed evidence of co-infections with different
Mycoplasma species, Chlamydia pneumoniae (Odds Ratio = 8.6, 95% CL 1.0-71.1, P<0.01) and/or active Human Herpes
Virus-6 (HHV-6) (Odds Ratio = 4.5, 95% CL 2.0-10.2, P<0.001). We found that 8% of the CFS patients showed evi-
dence of C. pneumoniae and 31% of active HHV-6 infections. 11,79 In a separate study we found that a sizable percentage
of CFS/ME patients were infected with Borrelia burgdorferi, and therefore, they were also Lyme Disease (LD) patients.80
8. Lyme Disease (LD)
Lyme Disease (LD) is the most common tick-borne disease in North America. First described in Southeastern Connecti-
cut in 1975, the infection is caused by a tick bite and the entry of the spiral-shaped spriochete Borrelia burgdorferi and
other co-infections.82 Borrelia b. and its co-infections have been carried into new habitats by a variety of ticks and their
vectors. After incubation for a few days to a month, the Borrelia spriochete and co-infections migrate through the subcu-
taneous tissues into the lymph and blood where they can travel to near and distant host sites.83 Transplacental transmis-
sion of Borrelia b. and co-infections can occur in pregnant animals, including humans, and blood-borne transmission in
humans by blood transfusion is likely but unproven. The tick-borne LD co-infections can and usually do appear clinically
at the same time.
Since the signs and symptoms of LD overlap with other chronic conditions, LD patients are often diagnosed with
other illnesses, such as CFS/ME or Rheumatoid Arthritis. However, many patients with LD fail to receive an adequate
diagnosis for years, and during this period ineffective treatments may contribute to the refractory nature of the disease.
About one-third of LD cases start with the appearance of a round, red, bulls-eye skin rash (erythema migrans) at
the site of the tick bite, usually within 3-30 days.83 Within days to weeks mild flu-like symptoms can occur that include
shaking chills, intermittent fevers and local lymph node swelling. After this localized phase, which can last weeks to
months, the infection(s) can spread to other sites (disseminated disease), and patients then show malaise, fatigue, fever
and chills, headaches, stiff neck, facial nerve palsies (Bell’s palsy) and muscle and joint pain and other signs/symptoms.83
LD can eventually become persistent or chronic and involve the central and peripheral nervous systems as well as
ophthalmic, cardiac, musculoskeletal and internal organ invasion. At this late chronic stage Rheumatoid Arthritis, neuro-
logical impairment with memory and cognitive loss, cardiac problems (mycocarditis, endocarditis causing palpitations,
pain, bradycardia, etc.) and severe chronic fatigue are often apparent.84,85 The late chronic phase of the disease usually
overlaps with other chronic conditions, such as CFS/ME, FMS, Rheumatoid Arthritis, among others, causing confusion in
the diagnosis and treatment of the chronic phase in LD patients.80.85 Some contend that this late phase is not even related
to LD, resulting in failure to successfully identify and treat the chronic condition.86
The involvement of co-infections in causing chronic signs/symptoms in LD patients has not been carefully inves-
tigated; however, such infections on their own have been shown to produce comparable signs/symptoms. Diagnostic labo-
ratory testing for LD at various clinical stages is, unfortunately, not full-proof, and experts often use a checklist of signs
and symptoms and potential exposures, along with multiple laboratory tests to diagnose LD.86 The laboratory tests used
for LD diagnosis include: detection of Borrelia b. surface antigens by enzyme-linked immunoassay (EIA), immunofluo-
rescent assay (IFA), and Western immunoblot of Borrelia proteins.86 Alternatively, polymerase chain reaction (PCR) for
Borrelia DNA has been used to detect the DNA of the intact organism in blood.85 A true-positive test result usually con-
sists of more than one positive test from the above list, often EIA followed by Western imunoblot.87 The problem with
these tests is that they are blood tests that require the presence of antibodies or Borrelia proteins in the blood, or they are
dependent on the spirochete and thus its DNA being present in the blood (PCR).
0%
20
40
60
80%
Lyme Patients Contain Various Microbial Species
Mycoplasma
all species
45-
70%
Borrelia
burgdorferi
Bartonella
all species
Chlamydia
pneumoniae
Prof. G. L. Nicolson
Institute for Molecular Medicine
25-40%
<10%
Ehrlichia
all species
Babesia
all species
8-20%
10-35%
Figure 6. The incidence of various bacterial co-infections in 100 patients with Lyme Disease. The bars indicate the range of values
found in various laboratories. Incidence determined by serology, Western blot and PCR tests of blood.
We80,85 and others88 have found that the most common co-infection found with Borrelia b. are various species of
Mycoplasma (Fig. 6). Approximately 50-70% of LD patients also have mycoplasmal co-infections (M. fermentans > M.
pneumoniae, M. hominis > M. penetrans, other species). In some cases multiple mycoplasmal infections are present in
LD patients.80 The presence of mycoplasmal infections complicates the diagnosis and treatment of LD, and some of the
generalized signs/symptoms found in Borrelia-positive patients are also found in mycoplasma-positive patients. Like the
Borrelia b. spirochete, Mycoplasma species are found at intracellular locations in various tissues and are only rarely found
free in the blood. This can make detection difficult, and in some patients the appearance of Borrelia b. and various My-
coplasmas in their white blood cells can be cyclic.
Other LD coinfections include: Ehrlichia species, Bartonella species and Babesia species.89 Ehrlichia species are
small, gram-negative, pleomorphic, obligate intracellular infections similar to mycoplasmas in their structures, intracellu-
lar locations and resulting signs/symptoms.90 The other common bacterial co-infection is caused by Bartonella spp.,91 and
this co-infection (along with Mycoplasma spp.) appears to be one of the most common tick-borne co-infections found
with Borrelia burgdorferi.91 Bartonella spp., such as Bartonella henselae, which also causes cat-scratch disease,92 is of-
ten found in neurological cases of Lyme Disease.91 A non-bacterial co-infection found with Borrelia burgdorferi is the
intracellular protozoan Babesia species.93 There are over 100 species of the genus Babesia, but most Lyme Disease co-
infections in humans in North America are caused by Babesia microti.94 About 10-40% of cases of LD show Babesia co-
infections (Fig. 6).
The combination of Borrelia, Mycoplasma and Babesia infections can be lethal in some patients (about 7% of pa-
tients can have disseminated intravascular coagulation, acute respiratory distress syndrome and heart failure), but the ma-
jority of LD patients with Babesia spp. have the chronic form of the infection. These patients can show mild to severe
hemolytic anemia (probably correlating with the protozoan colonization of erythrocytes, which can be seen by experi-
enced individuals in blood smears) and a normal to slightly depressed leukocyte count. However, this is usually not seen
in patients who have progressed to the chronic phase of the disease. 93 The chronic form of LD with CNS invasion is usu-
ally called neuroborreliosis, and this can be a fetal disease.84
8. Final Comment
Chronic illness patients are at risk for a variety of opportunistic infections, including bacterial, viral and fungal infections.
These can complicate diagnosis and treatment, and they may be a particular problem in the late, chronic phase of the dis-
ease. Late-stage patients with neurological manifestations, meningitis, encephalitis, peripheral neuropathy or other signs
and symptoms may have complicated coinfections that are not recognized or treated by their physicians.
The neurological signs and symptoms in many, more likely most, chronic illness patients are usually due to sys-
temic chronic infections that penetrate the CNS. Such infections often follow acute or chronic heavy metal, chemical,
biological (viral, bacterial, fungal infections) exposures or other environmental insults or even multiple vaccines that have
the potential to suppress the immune system and allow opportunistic infections to take hold. These illnesses generally
evolve slowly over time in a multi-step process that likely require genetic susceptibility along with multiple toxic expo-
sures. Because of this, they are particularly difficult to treat using single modality approaches. Importantly, if complex,
chronic infections are ignored or left untreated in these illnesses, it is unlikely that recovery will follow. We have also
stressed that integrative approaches to therapy offer the most realistic chance for patients to eventually recover.
An integrative approach to the treatment of the complex, slow-growing intracellular infections found in a variety
of chronic diseases requires long-term treatment with antibiotics and other antimicrobials, and it also requires dietary sup-
plementation to restore normal homeostasis.85,94 For example, most if not all chronic illness patients require dietary sup-
plementation with vitamins, minerals, amino acids, lipids and other natural supplements.85,94-97 These are necessary to re-
store intracellular functions that are damaged by infections but also damaged by environmental stresses, heavy metals,
chemicals and other contaminating substances. Improving or restoring normal neurological, immunological and hormonal
functions to patients with complex neurodegenerative and other chronic diseases remain difficult and important goals.
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