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 neurodegenerative and behavioral neurological conditions, such as Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Alzheimer's 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, Borrelia 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 illnesses where neurological manifestations are often found, such as Chronic Fatigue Syndrome/Myalgic Encephlomyopathy (CFS/ME), Fibromyalgia Syndrome (FMS), Lyme disease (LD), 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.

Introduction
Neurodegenerative diseases are chronic degenerative diseases of the central nervous system (CNS) that often cause dementia 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 diseases are characterized by molecular changes in nerve cells that result in nerve cell degeneration and, ultimately, nerve dysfunction and cell death, resulting in neurological signs and symptoms and, eventually, 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, deletions, or amplifications.¹ In addition to genetic changes and changes in gene expression, it is thought that nutritional deficiencies, 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 involved in various neurodegenerative diseases.^1-5 One of the biochemical changes found in essentially all neurological degenerative 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 in 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 transfer from peripheral nerves.⁶ Cell wall-deficient bacteria, principally species of Mycoplasma, Chlamydia, Coxiella, Brucella, Borrelia, among others, are candidate infectious agents that may play an important role in neurodegenerative diseases.⁸ Such infections may also cause disease progression, and since they are usually systemic, they could affect the immune system and other organ systems, resulting in systemic signs and symptoms.⁹

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 separated 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.

Amyotrophic Lateral Sclerosis
Amyotrophic Lateral Sclerosis (ALS) is an adult-onset, idiopathic, 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 pathological 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 veterans (from three nations) diagnosed with ALS 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 US, Canada, and Great Britain, we also found blood Mycoplasmal infections. Of the Mycoplasma-positive civilian patients who were further tested for M. penetrans, M. fermentans, M. hominis, and M. pneumoniae, most were positive 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 Figure 1. The other type of infection that is commonly found in ALS is Lyme Borrelia burgdorferi (Figure 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

Figure 1: Percent incidence of systemic bacterial and viral infections in 46 patients with Amyotrophic Lateral Sclerosis. 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 (Figure 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) accumulation 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.

Multiple Sclerosis
Multiple Sclerosis (MS) is a disease of the nerves of the central nervous system, and it can occur in young as well as older people. The nerves in various parts of the brain are covered by a protective insulation containing the protein myelin and other proteins embedded in a lipid sheath so that the electrical impulses that cause nerve conduction are protected. In MS, inflammation and the presence of autoimmune antibodies against myelin and other nerve cell antigens cause 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 inflammation 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 Epidemiological and twin studies suggest that MS is acquired not inherited. Since more than 90% of MS patients show immunological and cytokine characteristic of infection, MS patients have been examined for various viral and bacterial infections. 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, Human Herpes Virus-6, 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 burgdorferi (Lyme disease), and other intracellular bacterial infections may also be involved in some MS cases (Figure 2). When these infections are released from cells, they contain host cell antigens in their exterior membranes, and these normal cell membrane antigens could stimulate autoimmune responses. Alternatively, the microorganisms may express antigens that mimic normal surface antigens.

Figure 2: Percent incidence of systemic bacterial and viral infections in 65 patients with Multiple Sclerosis. The results were determined by Western blot or PCR.

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 (Figure 2), suggesting 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.

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 different 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 Mycoplasma 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

Alzheimer's Disease
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 tangles may be due to genetic defects and resulting changes in the structure of Ab proteins, neurotoxicity caused by chemicals or other toxic events, inflammatory 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 considerable 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 bacteria 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 parenchyma.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 association 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 Borrelia 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 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 co-infections are found in AD, and an interesting relationship 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

Autistic Spectrum Disorders
Children with Autistic Spectrum Disorders (ASD), such as autism, attention deficit disorder, Asperger syndrome, etc., generally 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, communication, 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 structure. 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, 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 and heavy metal, chemical, and biological exposures, among others, and are probably different in each patient.^64,65 However, among ASD patients, there may be similarities in genetic defects and environmental exposures that are important in patient morbidity (sickness) or in illness progression. 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 progression.

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 commonly have been 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 for measles, mumps, and rubella (MMR) came into widespread use. In the US, children 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 six percent of commercial vaccines 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 nonspecific 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 three to 12 who were also diagnosed with autism or attention deficit hyperactivity disorder (ADHD), two disorders that fall under ASD.⁷³ In our study, 45 of 110 GWI patients or ~42% had Mycoplasmal infections (Figure 3), and almost 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 (Figure 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 six 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

Figure 3: Percentage incidence of Mycoplasmal infections in family members of verterans with Gulf War Illnesses. The results were determined by PCR.⁷⁰

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 members who had similar infections (6/50 or 12%) (Figure 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 species 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 10.0%), this was not significantly different from the incidence of Mycoplasmal infections in healthy control subjects (6/70 or 8.5%) (Figure 3).⁷⁰

The Mycoplasma species was also similar between GWI patients and their CFS/ME-symptomatic family members. 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 infections 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 three to 12 who were diagnosed with ASD. Many of these children had at least one parent with a chronic illness, and the most common diagnosis of adults or adolescents in the same family was CFS/ME or fibromyalgia syndrome. When the ASD patients 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. fermentans, the Central California group 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

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 patients for other infections (Figure 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 Ratio=5.6, P<0.01) and 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 infections (Odds Ratio=16.5, P<0.001).⁷⁵

Figure 4: Percentage incidence of bacterial and viral infections in 48 patients with Autistic Spectrum Disorders.⁷⁵ The range indicates results from laboratories. Incidence was determined by Western blot, serology, or PCR.

Chronic Fatigue Syndrome
Chronic fatigue syndrome (CFS/ME) 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 associated 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 infections.^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 (Figure 5), is likely to be important in determining the treatment strategies for these CFS/ME patients.^11,79-81

Figure 5: The incidence of various bacterial and viral co-infections in 100 patients with CFS/ME. The bars indicate the range of values 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 patients 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 Mycoplasma 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 HHV-6 (Odds Ratio = 4.5, 95% CL 2.0-10.2, P<0.001). We found that eight percent of the CFS patients showed evidence 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 patients.⁸⁰

Lyme Disease
Lyme disease (LD) is the most common tick-borne disease in North America. First described in Southeastern Connecticut in 1975, the infection is caused by a tick bite and the entry of the spiral-shaped spirochete 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 spirochete and co-infections migrate through the subcutaneous tissues into the lymph and blood where they can travel to near and distant host sites.⁸³ Transplacental transmission 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 three to 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, neurological 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 investigated; however, such infections on their own have been shown to produce comparable signs/symptoms. Diagnostic laboratory 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 the following: detection of Borrelia b. surface antigens by enzyme-linked immunoassay (EIA), immunofluorescent 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 consists of more than one positive test from the above list, often EIA followed by Western immunoblot.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).

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 seriology, Western blot and PCR tests of blood.

We^80,85 and others⁸⁸ have found that the most common co-infection found with Borrelia b. are various species of Mycoplasma (Figure 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 Mycoplasmas in their white blood cells can be cyclic.

Other LD co-infections 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, intracellular 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 often 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 (Figure 6).

The combination of Borrelia, Mycoplasma, and Babesia infections can be lethal in some patients (about seven percent of patients can have disseminated intravascular coagulation, acute respiratory distress syndrome, and heart failure), but the majority 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 experienced 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 usually called neuroborreliosis, and this can be a fatal disease.⁸⁴

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 disease. Late-stage patients with neurological manifestations, meningitis, encephalitis, peripheral neuropathy, or other signs and symptoms may have complicated co-infections 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 systemic 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 requires genetic susceptibility along with multiple toxic exposures. 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 supplementation to restore normal homeostasis.^85,94 For example, most if not all chronic illness patients require dietary supplementation with vitamins, minerals, amino acids, lipids, and other natural supplements.^85,94-97 These are necessary to restore intracellular functions that are damaged by infections and 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.

The Institute for Molecular Medicine
16371 Gothard Street H
Huntington Beach, California 92647
949-715-5978; Fax: 714 596-3791;
gnicolson@immed.org
www.immed.org

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