Rheumatoid Arthritis

Clinical Practice of Alternative Medicine 2000;1(2):92-102.
Official journal of the American College of Advancement in Medicine

Diagnosis and Integrative Treatment of Intracellular Bacterial Infections in Chronic Fatigue and Fibromyalgia Syndromes, Gulf War Illness,
Rheumatoid Arthritis and other Chronic Illnesses
Garth L. Nicolson,* PhD, Marwan Y. Nasralla,*+ PhD, A. Robert Franco,‡ MD,
Nancy L. Nicolson,* PhD, Robert Erwin,* MD, Richard Ngwenya,† MD, and Paul A. Berns,* MD

* The Institute for Molecular Medicine, Huntington Beach, CA 92649 USA,
+ International Molecular Diagnostics, Inc., Huntington Beach, CA 92649 USA
‡ Arthritis Center of Riverside, Riverside, CA 92501 USA,
† James Mobb Immune Enhancement, Harare, Zimbabwe

Address correspondence to: Prof. Garth L. Nicolson, The Institute for Molecular Medicine, 16371 Gothard St. H, Huntington Beach, CA 92647. Tel: 714-596-6636; Fax: 714-596-3791; Website: www.immed.org; Email: gnicolson@immed.org.

ABSTRACT

Bacterial and viral infections are associated with many chronic illnesses as causative agents, cofactors or more likely as opportunistic infections in immune suppressed individuals. The prevalence of invasive pathogenic Mycoplasma species infections (and possibly other bacterial infections, such as Chlamydia, Borrelia, etc.) in patients with Chronic Fatigue Syndrome, Fibromyalgia Syndrome, Gulf War Illness, Rheumatoid Arthritis and other chronic illnesses was significantly higher than in healthy controls. When we examined chronic illness patients for multiple Mycoplasma species infections, we found that almost all patients had multiple intracellular infections, suggesting that multiple bacterial infections commonly occur in certain chronic illness patients. These patients generally respond to particular antibiotics if administered long-term, but an important part of their recovery involves nutritional supplementation with appropriate vitamins, minerals, immune enhancement and other supplements. Nutraceuticals appear to be necessary for recovery and maintenance of a strong immune system. In addition, patients should be removed from potentially immune-depressing drugs, such as some antidepressants, to allow recovery of their immune systems. Other chronic infections (viral), may also be involved in various chronic fatigue illnesses with or without mycoplasmal and other bacterial infections, and these multiple infections could be important in causing patient morbidity and resulting difficulties in treating these illnesses.

INTRODUCTION

Most if not all debilitating chronic illnesses are characterized by the presence of chronic fatigue (1), the most commonly reported medical complaint of all patients seeking medical care (2). The fatigue syndromes, such as Chronic Fatigue Syndrome (CFS or Myalgic Encephalomyelitis [ME]), Fibromyalgia Syndrome (FMS) and Gulf War Illnesses (GWI), share many complex, multi-organ signs and symptoms (3-6), including immune system abnormalities (7), but are distinguishable as separate syndromes that have muscle and overall fatigue as major signs. These syndromes usually have overlapping signs and symptoms, including muscle pain, chronic fatigue, headaches, memory loss, nausea, gastrointestinal problems, joint pain, vision problems, breathing problems, depression, low grade fevers, skin disorders, tissue swelling, chemical sensitivities, among others (5, 6, 8). Because of the complex nature of these illnesses, many patients are often diagnosed with multiple syndromes, and their potential to recover from their chronic illness is usually poor at best.

Chronic illness patients usually have cognitive problems, such as short-term memory loss, depression, difficulty concentrating and psychological problems that can result in practitioners diagnosing chronic illness patients with somatoform disorders rather than organic problems (6, 8). Thus due to the lack of definitive laboratory or clinical tests that could identify the cause(s) of chronic illnesses, such disorders are thought to be caused for the most part by psychological stressors. In fact, emotional stress is often an important factor in somatoform disorders, and stress itself can have many effects on the hormonal and immune systems that could be detrimental in virtually any chronic illness (9). But we feel strongly that stress alone is unlikely to cause most of the chronic illnesses discussed here, the most classic being GWI (6, 8), where battlefield stress was promoted as the cause of the illness (10). GWI patients are often diagnosed with Post Traumatic Stress Disorder (PTSD), but the evidence that stress or PTSD is the source of GWI is based on the assumption that veterans must have suffered from stress by virtue of the stressful environment in which they found themselves during the Gulf War (10). The notion that stress is the major factor in GWI or indeed in other chronic illnesses, we feel, is not supported by most evidence that suggests that these illnesses were caused by toxic exposures (10, 11).

There is growing awareness that the chronic fatigue illnesses, such as CFS/ME, FMS, GWI and certain autoimmune illnesses, such as Rheumatoid Arthritis (RA), among others, can have an infectious nature that is either responsible (causative) for the illness, a cofactor for the illness (required but not the only causative factor) or more likely appears as an opportunistic infection(s) responsible for aggravating or causing patient morbidity (8, 11, 12). There are several reasons for this notion, including the nonrandom or clustered appearance of the illnesses, often in immediate family members, the course and signs/symptoms of the illnesses and their responses to therapies based on treatments directed at infectious agents and enhancement of immune responses. Most chronic fatigue illnesses are difficult to treat and do not have effective therapies, and these patients rarely recover from their illnesses (11), causing in some cases catastrophic economic problems. Here we will discuss methods for diagnosing chronic infections in patients with CFS/ME, FMS, GWI, RA and other chronic illnesses and offer some suggestions for appropriate treatments directed at some of the chronic infections that play important roles in these illnesses.

SIMILAR SIGNS AND SYMPTOMS OF CHRONIC ILLNESSES

Most chronic illnesses have complex but relatively nonspecific signs and symptoms that are not characteristic for a particular disease. However, other chronic illnesses, such as RA, are well established in their diagnostic profiles (13, 14). One difference between some of the most common chronic illnesses appears to be in the severity of particular signs and symptoms. For example, in CFS/ME essentially all patients complain of chronic fatigue and joint pain, stiffness and soreness, whereas in FMS essentially all patients complain of muscle and overall pain, soreness and weakness. But when secondary signs and symptoms of these chronic illnesses are compared, they look very similar (6, 8). For the most part, the signs/symptom profiles of CFS/ME, FMS, GWI illnesses are similar (Fig. 1). Thus the chronic illnesses under discussion here have overlapping signs and symptoms, suggesting that these illnesses may be related (8). In addition, CFS/ME, FMS and GWI patients often show increased sensitivities to various environmental irritants and chemicals and enhanced allergic responses, suggesting that their immune systems are, at least in part, dysfunctional. This is supported by laboratory studies on the natural immune and other immunological abnormalities in chronic illness patients (7).

The overlapping signs and symptoms of many chronic illness patients are easily documented. For example, the patient signs/symptoms data presented in Figure 1 were obtained using patient Illness Survey Forms to determine common signs and symptoms at the time when blood was drawn from patients for analysis. In this figure the intensity of approximately 120 patient signs and symptoms prior to and after onset of illness were recorded on a 10-point rank scale (0-10, extreme). The data were then arranged into 29 different signs and symptoms groups and were considered positive if the average value after onset of illness was two or more points higher than prior to the onset of illness. The CFS/ME and FMS patients had complex signs and symptoms that were similar to those reported for GWI, and the presence of rheumatic signs and symptoms in each of these disorders indicates that there are also some similarities to RA (13-15) (Fig. 1). Some differences were noted, however, when patients with chronic illnesses without evidence of intracellular bacterial infections were compared to the above groups (Fig. 1). The data suggest that patients with intracellular bacterial infections have more complex clinical signs/symptoms.

In our signs/symptom analyses it was not unusual to find immediate family members who displayed similar chronic signs and symptoms. For example, we found that the spouses and children of GWI patients often slowly developed chronic illnesses with signs and symptoms similar to GWI, but only some time after the return home of veterans who developed GWI (8, 10, 11). That these civilian patients contracted their illnesses from chronically ill family members with GWI was a likely explanation (8) that was supported by the finding of similar chronic infections in these families (8, 11).

CHRONIC INFECTIONS AND MORBIDITY IN CFS/ME, FMS AND GWI

Although chronic illnesses have been known in the medical literature for years, most patients with CFS/ME, FMS, GWI and in some cases RA have had few treatment options. This is probably due to the fact that the underlying causes of most chronic illnesses are unknown and treatments have been mainly palliative or supportive. Even if the causes or triggering events in chronic illnesses are not understood, these illnesses may show similarities in their progression; that is, they could have different initial causes or triggers but similar secondary events that result in progression (8, 11). We have proposed that the secondary event(s) could be opportunistic viral and/or bacterial infections that cause significant morbidity and illness progression (10-12). With time these secondary events may evolve or progress to be important or even dominant factor(s) in determining overall signs/symptoms and treatment strategies.

Since indirect evidence suggests the infectious nature in at least certain subsets of chronic illness patients (8, 11, 12), we have been examining chronic illness patients for pathogens that could explain, at least in part, their complex signs and symptoms. One type of infection that could fulfill the criteria of association with a wide range of chronic illness signs and symptoms are certain microorganisms of the class Mollicutes (8, 11, 12). This is a class of small bacteria, lacking cell walls and genetics for lipid and other macromolecule synthesis pathways. It is primarily composed of Mycoplasmas, and although most species are nonpathogenic, some pathogenic Mycoplasma species are capable of invading several types of human cells and tissues and are associated with a wide variety of human diseases (11, 15-19).

Are pathogenic Mycoplasma species and other intracellular bacteria (Chlamydia, Borrelia, etc.) associated with chronic illnesses such as CFS/ME, FMS, GWI and RA? We (6, 8, 11, 15, 17-19, 24) and others (20-23) have examined chronic illness patients for the presence of mycoplasmal blood infections and have found a strong association with the presence of chronic illnesses. In our studies the clinical diagnosis of these disorders was obtained from referring physicians according to the patients’ major signs and symptoms. Blood was collected, shipped over night at 4°C and processed immediately for Nucleoprotein Gene Tracking (NPGT) after isolation of blood leukocyte nuclei (17, 18) or Polymerase Chain Reaction (PCR) after purification of blood leukocyte DNA using a Chelex procedure (6, 8, 15, 19). These procedures are very sensitive and specific and can detect down to a few copies of intracellular bacteria in a blood sample. The sensitivity and specificity of the methods were determined by examining serial dilutions of purified DNA of M. fermentans, M. pneumoniae, M. penetrans and M. hominis in blood samples. Amounts as low as 1-10 fg of purified microorganism DNA were routinely detectable. Using PCR the amplification with the appropriate primers produced the expected fragment size in all tested species, which was confirmed by hybridization with an inner probe or DNA sequencing to confirm the sequence of the PCR product.
We used the NPGT and the PCR procedures to examine chronic illness patients for Mycoplasma species and Chlamydia species infections. For example, using NPGT to analyze the blood leukocytes of GWI patients we found that 91/200 (~45%) were positive for mycoplasmal infections. In contrast, in nondeployed, healthy adults the incidence of mycoplasmal infections was 4/62 (~6%) (17, 18). Similarly, others have more recently used PCR to examine GWI patients and found that 55% were positive for Mycoplasma species and 36% were found to have M. fermentans infections (23). The slight difference in percentage of positive patients is probably due to the differences in sensitivities of these two methods. Using PCR procedures 52-63% of CFS/ME and FMS patients (n~1,000) had mycoplasmal infections (6, 19-24), whereas only 6-15% of controls (n~450) tested positive.

An important observation was that patients with chronic illnesses that test positive for mycoplasmal infections usually have multiple infections. When we examined mycoplasma-positive CFS/ME and FMS patients (~60% of such patients are usually mycoplasma-positive) for the presence of M. fermentans, M. pneumoniae, M. penetrans, M. hominis infections, multiple infections were found in the majority of approximately 100 patients (19). CFS/ME/FMS patients had two (>30%) or three (>20%) species of mycoplasmal infections, but only when one of the species was M. fermentans or M. pneumoniae (19). We also found higher score values for increases in the severity of signs and symptoms after onset of illness in CFS/ME/FMS patients with multiple infections. Also, CFS/FMS patients with multiple mycoplasmal infections generally had a longer history of illness, suggesting that patients may have contracted additional infections during their illness (19). Most of these patients also show evidence of various viral and Chlamydia species infections. Thus it is likely that most CFS/ME and FMS patients have multiple bacterial and viral infections.

DIAGNOSIS OF CHRONIC INFECTIONS IN ARTHRITIS PATIENTS

The causes of rheumatic diseases are for the most part unknown, but RA and other autoimmune diseases could be triggered or more likely exacerbated by infectious agents (25). In some animal species infection by certain Mycoplasma species can result in remarkable clinical and pathological similarities to RA and other rheumatic diseases. Aerobic and anaerobic intestinal bacteria, viruses and mycoplasmas have all been proposed as possible agents in the etiology of RA (25-30), and there has been increasing evidence that mycoplasmas may play a role in the initiation or more likely progression of RA (13, 15, 30-32). Mycoplasmas have been proposed to interact nonspecifically with B-lymphocytes, resulting in modulation of immunity, autoimmune reactions and promotion of rheumatic diseases (31), and mycoplasmas have been found in the joint tissues of patients with rheumatic diseases, suggesting their pathogenic involvement in these and other chronic illnesses (29).

Using PCR Mycoplasma species are commonly found in RA patients’ blood. For example, when Haier et al. (15) and Vojdani and Franco (23) examined RA patients’ blood leukocytes for the presence of mycoplasmas, they found that approximately one-half were infected with various species of mycoplasmas. The most common species found was M. fermentans, followed by M. hominis, M. pneumoniae and finally M. penetrans (15, 23). Similar to what we reported in CFS/FMS patients (19), there was a high percentage of multiple mycoplasmal infections in RA patients when one of the species was M. fermentans (15).

Mycoplasma species and other intracellular pathaogenic bacteria could be important factors or cofactors in the development of inflammatory responses in rheumatic diseases and for progression of RA. As an example of the possible role of Mycoplasma species in rheumatic diseases, M. arthritidis infections in animals can trigger and exacerbate autoimmune arthritis in animal models of RA (32, 33). M. arthritidis can also suppress immune cells and release substances that act on polymorphonuclear granulocytes, such as oxygen radicals, chemotactic factors and other substances (33). Mycoplasmal infections can increase pro-inflammatory cytokines, such as Interleukin-1, -2 and –6 (34), suggesting that they are involved in the development and possibly progression of rheumatic diseases such as RA. In addition, mycoplasmas have been detected in the synovial fluid of RA patients’ joints (29).

A variety of microorganisms have been under investigation as cofactors or causative agents in rheumatic diseases (8, 15, 25, 26). The discovery of EB virus (27) and cytomegalovirus (28) in the cells of the synovial lining in RA patients suggested their involvement in RA, possibly as cofactors. There are a number of bacteria and viruses that are candidates in the induction or progression of RA (15, 25, 26). In support of a bacterial involvement in RA, antibiotics like minocycline can alleviate the clinical signs and symptoms of RA (Table 1) (35). This and similar drugs are likely suppressing infections of sensitive microorganisms like mycoplasmas, although certain antibiotics could also cause other effects in susceptible patients.

MYCOPLASMAL INFECTIONS IN OTHER CHRONIC ILLNESSES

Mycoplasmas have been associated with the progression of immunosuppressive diseases, such as HIV-AIDS (36). These infections have also been associated with certain lethal human diseases, such as an acute fatal illness found with M. fermentans infections in non-AIDS patients (37). Importantly, mycoplasmal infections are now thought to be a major source of morbidity in HIV-AIDS (38). Expanding further on this, Blanchard and Montagnier (38) have proposed that certain mycoplasmas like M. fermentans are important cofactors in the progression of HIV-AIDS, accelerating disease progression and accounting, in part, for the increased susceptibility of AIDS patients to increased viral replication and additional opportunistic infections. Since most studies on the incidence of mycoplasmal infections in HIV-AIDS patients have employed relatively insensitive tests, it is likely that the actual prevalence of mycoplasmal infections in HIV-AIDS patients is much greater than previously thought and may be associated with a rapid fatal course of the disease. For example, in HIV-AIDS mycoplasmas like M. fermentans can cause renal and CNS complications (39), and mycoplasmas have been found in various tissues, such as the respiratory epithelial cells of AIDS patients (40). Other species of mycoplasmas have been found in AIDS patients where they have also been associated with disease progression (41), and it is likely that several viral and bacterial infections are involved in the progression of this disease. In addition to immune suppression, some of this increased pathogenecity may be the result of mycoplasma-induced host cell membrane damage from toxic oxygenated products released from intracellular bacteria (42). Also, mycoplasmas may regulate HIV-1 virus replication. Interestingly, HIV-LTR-dependent gene expression can be regulated by the presence of certain pathogenic mycoplasmas (43).

There is some preliminary evidence that mycoplasmal and other infections are associated with various autoimmune diseases. For example, in some mycoplasma-positive GWI cases some of the signs and symptoms of Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), Lupus, Graves’ Disease and other complex autoimmune diseases have been seen. Such usually rare autoimmune responses are consistent with certain chronic infections, such as mycoplasmal infections, that penetrate into nerve cells, synovial cells and other cell types and probably stimulate autoimmune responses by their own or host antigens. Thus the autoimmune signs and symptoms in these patients could be the result of intracellular pathogens, such as mycoplasmas, escaping from cellular compartments and incorporating into their own structures pieces of host cell membranes that contain important host antigens that can trigger autoimmune responses. Alternatively, mycoplasma surface components, sometimes called ‘superantigens,’ may directly stimulate autoimmune responses (44). Perhaps the most important event, the molecular mimicry of host antigens by mycoplasma surface components, may explain, in part, their ability to stimulate autoimmune responses (45).

Pulmonary infections are often seen in chronic illness patients. For example, asthma, airway inflammation, chronic pneumonia and other respiratory diseases are known to be associated with mycoplasmal infections (46). It has been noted that M. pneumoniae is a common cause of upper respiratory infections (47), and severe asthma is frequently associated with mycoplasmal and other infections (48). Chronic illness patients with respiratory signs and symptoms usually have bacterial infections.

An emerging area of interest is the possible involvement of chronic infections in a variety of coronary conditions. Cardiopathies can be caused by chronic Mycoplasma species (49) and Chlamydia species (50) infections, resulting in myocarditis, endocarditis, pericarditis and other types of infections. These cardiac infections are often due to Mycoplasma species, Chlamydia species and possibly other intracellular bacteria and other infectious agents, and they are emerging agents in coronary diseases.

Mycoplasmal infections are also associated with a variety of miscellaneous illnesses, such as M. hominis infections in patients with hypogamma-globulinemia (30), and M. genitalium infections in nongonococcal urethritis patients (51). Mycoplasmas can exist in the oral cavity and gut as normal flora, but when they penetrate into the blood and tissues, they may be able to cause or promote a variety of acute or chronic illnesses. These cell-penetrating species, such as M. penetrans, M. fermentans, M. hominis and M. pirum, among others, can cause infections that result in complex systemic signs and symptoms. Mycoplasmal infections can also cause synergism with other infectious agents. Similar types of chronic infections caused by cell-invasive Chlamydia, Brucella, Coxiella or Borriela species may also be present either as single agents or as complex, multiple infections in many chronic illnesses (8, 11).

CONVENTIONAL TREATMENT OF CHRONIC BACTERIAL INFECTIONS

Once chronic intracellular bacterial infections, such as Mycoplasma species infections, have been identified in the blood of subsets of CFS/ME, FMS, GWI, RA and other chronic illness patients, they can be treated using conventional and alternative approaches. Appropriate treatment with antibiotics should result in patient improvement and even recovery, and this has been found in most but not all chronic illness patients (8, 11, 17, 18, 52-54) (Table 1). The recovery is usually slow and gradual after an initial period of Herxheimer and other adverse reactions that make patients temporarily more symptomatic. This period can last for several weeks. The recommended treatments for mycoplasmal blood infections are usually long-term antibiotic therapy, usually multiple 6-week cycles of doxycycline (200-300 mg/day), ciprofloxacin (1,500 mg/day), azithromycin (250-500 mg/day) or clarithromycin (750-1,000 mg/day), among others (53). Multiple 6-week cycles are required, because few patients recover after only a few cycles of antibiotics. This is probably due to the intracellular locations of mycoplasmas like M. fermentans and M. penetrans or other bacteria, such as Chlamydia species, the slow-growing nature of these infections, their inherent insensitivity to most antibiotics and the persistence of the infections in metabolically inactive forms. For most patients, treatment must be continuous for at least 6 months, followed by additional 6-week cycles of antibiotics, if necessary. Some treat these infections by administration of antibiotics every other day, and some recommend daily dosing. Due to poor gastrointestinal absorption in certain patients or the acuteness of signs and symptoms, intravenous therapy has been used for a few weeks, followed by oral antibiotics. Most patients cannot tolerate intravenous antibiotics for more than a few weeks or complications can then occur, so follow-on therapy with oral antibiotics is necessary.

Can antibiotic therapy be successful in treating intracellular bacterial infections often found in chronic illness patients? Yes, but antibiotics should not be used solely or exclusively to treat intracellular bacterial infections. They have proven successful for many if not most patients; however, many patients eventually fail on antibiotic therapy alone. For example, of 87 GWI patients that tested positive for mycoplasmal infections, all patients relapsed after the first 6-week cycle of antibiotic therapy, but after up to 6 cycles of therapy 69/87 previously mycoplasma-positive patients recovered and returned to active duty (17, 18). Since few patients recovered within 6 months of antibiotic therapy, as discussed above, this is now the minimal recommendation of antibiotic treatment (54). These were relatively young patients (most <25 years of age) that were healthy before their illness, and this could have played a role in their higher response rates compared to civilians with chronic illnesses which tend to be on the average older and not as healthy. The clinical responses that were seen in patients were not due to placebo effects, because administration of some antibiotics, such as penicillins, resulted in patients becoming more not less symptomatic, and they were not due to immunosuppressive effects that can occur with some of the recommended antibiotics. Interestingly, CFS/ME, FMS and GWI patients that slowly recovered after several cycles of antibiotics were generally less environmentally sensitive, suggesting that their immune systems were slowly returning to pre-illness states. If such patients had illnesses that were caused by psychological or psychiatric problems or solely by chemical or viral exposures, they should not have responded to the recommended antibiotics and slowly recovered. In addition, if such treatments were just reducing autoimmune responses, then patients should have immediately relapsed after the treatments were discontinued and they should not have responded to antibiotics that do not suppress immune systems.

Although the majority of GWI patients in these unblinded, initial studies responded to antibiotic therapy, the studies have been justifiably criticized for not being controlled, blinded clinical trials. In the case of GWI, large double-blinded, placebo-controlled studies have recently been initiated using doxycycline. In the case of RA, however, double-blinded, placebo-controlled antibiotic trials using minocycline have been successfully conducted. These trials show that the treatment of RA patients with minocycline is clinically effective and results in recovery of approximately one-half of an unselected group (not tested for chronic infections) of patients (Table 1) (35, 55). The reason for the incomplete responses among RA patients was probably due to the fact that only a portion of the patients under study probably had intracellular bacterial pathogens as their main clinical problem. Viruses and other pathogens may also play an important role in these patients, and minocycline would not be expected to have any effect on this type of infection.

Another less conventional approach to the treatment of chronic illness patients with intracellular bacterial infections is oxygen therapy. Hyperbaric oxygen, intravenous ozone and hydrogen peroxide have been used to treat anaerobic infections similar to the infections discussed here. Most patients with anaerobic infections respond to such therapy with at least temporary alleviation of signs and symptoms, but additional evidence is needed before one can conclude that such therapy results in sustained suppression of anaerobic infections, such as those caused by Mycoplasma and Chlamydia species. Since these therapies are mainly cytostatic not cytotoxic, they must be sustained for some period of time. However, the use of long-term protocols that include oxygen therapy is likely to prove useful, and it is certainly beneficial in patients who cannot tolerate antibiotics due to chemical sensitivities or other reasons. In some patients, alternating antibiotic and oxygen therapies may be useful, but this has not been done in a controlled study.

NUTRITIONAL SUPPLEMENTS FOR CHRONIC ILLNESS PATIENTS

For the therapy of chronic illness patients to be successful we believe that a comprehensive approach involving conventional and alternative therapies must be undertaken with each patient. An important part of chronic illness treatment programs should be the use of certain dietary supplements, particularly to boost and maintain immune systems (Table 2). In addition to treatments like antibiotics and antivirals, oxygen therapy and removal of toxic agents from the patients’ systems, nutritional supplementation should also be undertaken, especially in those patients with environmental toxic exposures (56). Although for the most part these alternative medical approaches have not been carefully evaluated in blinded trials, practitioners that have used them strongly support their usefulness.

Since chronic illness patients often have nutritional and other deficiencies, these should be corrected with the use of various supplements (Table 2) (53). For example, chronic illness patients are often depleted in vitamins B, C and E, among others, and certain minerals. Unfortunately, patients with chronic illnesses often have poor gastrointestinal absorption capacities. Therefore, relatively high doses of some vitamins must be used (vitamins C and E). Others, such as vitamin B complex, cannot be easily absorbed by the gastrointestinal systems of chronic illness patients, so sublingual or parentral natural B-complex vitamins (riboflavin, niacin, vitamin B-6, B-12 and pantothenic acid) should be substituted for oral preparations. General vitamins plus extra C, E, CoQ-10, beta-carotene, folic acid, bioflavoids and biotin are necessary. L- cysteine, L-tyrosine, L-glutamine, L-carnitine, malic acid and flaxseed or fish oils are reported by some to be useful. Certain minerals are also often depleted in these patients, such as zinc, magnesium, chromium and selenium, and these should be supplemented as well (Table 2) (56). One problem with providing supplements in a program that also uses antibiotics is that they cannot be taken at the same time of day as the antibiotics because they may inhibit antibiotic uptake or interfere with antibiotic transport. Another problem is the consumption of foods that can naturally suppress immune systems, such as processed sugar. We generally suggest that chronic illness patients undergoing therapy should make an effort to eliminate if possible sugar, alcohol, caffeine or other foods that may interfere with a patient’s immune system.

There are also other important considerations in patients undergoing antibiotic or antiviral therapy (53). Antibiotics deplete normal gut bacteria, which can result in over-growth of less desirable bacteria. To supplement bacteria in the gastrointestinal system live cultures of Lactobacillus acidophillus in tablets, capsules or powder are recommended. One product is a mixture of Lactobacillus acidophillus, Lactobacillus bifidus and other bacteria with FOS (fructoologosaccharides) to promote growth in the gastrointestinal system. Various commercial formulations of probiotics are available to replenish gastrointestinal bacteria that have been killed or suppressed by antibiotic therapy.

A number of natural remedies that boost the immune system can be useful in the therapy of chronic illnesses. Among these are whole lemon/olive extract drink or an extract of olive leaves with antioxidants, plant extracts or purified plant products or milk proteins, such as whey. These products are useful during or after antibiotic therapy has been completed. Although these products appear to help some patients, their clinical effectiveness in various chronic illness patients for the most part has not been carefully evaluated. An exception is a Chinese herbal formulation (Calm Colon; Samra) that has been tested for benefit in Irritable Bowel Syndrome. In a randomized, double-blinded, placebo-controlled clinical trial this formulation was found to be effective in reducing the symptoms of Irritable Bowel Syndrome (57). In some cases natural products are known to stimulate immune systems, as shown in various in vitro assays (58). They should be used by patients during therapy to boost immune systems and especially after antibiotic therapy in a maintenance program to prevent relapse of illness (53).

Traditional herbal supplements have proven useful in the treatment of chronic illness patients (57, 58), especially in a maintenance program to prevent relapse of illness. African and Chinese natural immune enhancers and cleansers can help to restore natural immunity and aid absorption. This is an often overlooked but nonetheless important recommendation (Table 2). Unfortunately, it is difficult to recommend specific supplements as useful in all patients, but many patients that have undergone more traditional antibiotic and/or antiviral therapies often relapse after the therapy is completed without continued support with dietary supplements. Some of the recommended products in Table 2 have mild natural antibiotic, antiviral and antifungal properties, so they can be useful in certain patients. Although these natural products are known to help many chronic illness patients, their clinical effectiveness in GWI/CFS/FMS/RA patients has not been carefully evaluated.

Another consideration is the elimination of drugs that might suppress immunity. We have recommended that patients be taken off antidepressants and other potentially immune-suppressing drugs. Some of these drugs are used to help alleviate certain signs and symptoms, but in our opinion they can interfere with therapy, and they should be gradually reduced or eliminated.

CONCLUSIONS

We have proposed that chronic infections, especially multiple chronic bacterial and viral infections, are an appropriate explanation for much of the morbidity seen in rather large subsets of CFS/ME, FMS, GWI and RA patients, and in a variety of other chronic illnesses. Not every patient, however, will have this as a diagnostic explanation or have the same types of chronic infections. These infections need not be the triggering factor or cause of chronic illnesses and are probably more important in causing progression of disease. Nonetheless, chronic infections may cause most of the morbidity seen in these patients, and selective therapy of chronic infections supports this view. Additional research will be necessary to clarify the role of multiple infections in chronic diseases, but these patients should benefit from appropriate antibiotic, antiviral and nutraceutical therapies that alleviate morbidity. Additional controlled studies should be performed to determine the clinical effectiveness of alternative therapies and nutritional supplements in treating chronic illnesses.

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FIGURE LEGENDS

Figure 1A and 1B. Incidence of increase in severity of signs and symptoms in 300 chronic illness patients. Severity of illness was scored using 118 signs and symptoms on a 10-point scale (0, none; 10 extreme) prior to and after the onset of illness. Scores were placed into 29 categories containing 3-9 signs/symptoms and were recorded as the sum of differences between values before and after onset of illness divided by the number of questions in the category. Changes in score values of 2 or more points were considered relevant. Patient groups were: CFS, FMS, GWI or chronic illness patients without evidence of bacterial infection. Asterisk (*) indicates score = 0.

G. L. Nicolson,* PhD, M. Y. Nasralla,* PhD, A. R. Franco,* MD, K. De Meirleir,+ MD, N. L. Nicolson,* PhD, R. Ngwenya,t MD and J. Haier,* MD, PhD

* The Institute for Molecular Medicine, Huntington Beach, CA 92649 USA,
* Arthritis Center of Riverside, Riverside, CA 92501 USA,
^Internal Medicine, Free University of Brussels, 1090 Brussels, Belgium and t James Mobb Immune Enhancement, Harare, Zimbabwe

Address correspondence to: Prof. Garth L. Nicolson, The Institute for Molecular Medicine, 15162 Triton Lane, Huntington Beach, CA 92649
(Fax: 714-379-2083; Email: gnicimm@ix.netcom.com)

SUMMARY. Bacterial and viral infections are associated with several fatigue illnesses, including Chronic Fatigue Syndrome (CFS), Fibromyalgia Syndrome (FMS), Gulf War Illnesses (GWI) and Rheumatoid Arthritis (RA), as causative agents, cofactors or opportunistic infections. We and others have looked for the presence of invasive pathogenic mycoplasmal infections in patients with CFS, FMS, GWI and RA and have found significantly more mycoplasmal infections in CFS, FMS, GWI and RA patients than in healthy controls. Most patients had multiple mycoplasmal infections (more than one species). Patients with chronic fatigue as a major sign often have different clinical diagnoses but display overlapping signs/symptoms similar to many of those found in CFS/FMS. When a chronic fatigue illness, such as GWI, spreads to immediate family members, they present with similar signs/symptoms and mycoplasmal infections. CFS/FMS/GWI patients with mycoplasmal infections generally respond to particular antibiotics (doxycycline, minocycline, ciprofloxacin, azithromycin and clarithromycin), and their long-term administration plus nutritional support, immune enhancement and other supplements appear to be necessary for recovery. Examination of the efficacy of antibiotics in recovery of chronic illness patients reveals that the majority of mycoplasma-positive patients respond and many eventually recover. Other chronic infections, such as viral infections, may also be involved in various chronic fatigue illnesses with or without mycoplasmal and other bacterial infections, and these multiple infections could be important in causing patient morbidity and difficulties in treating these illnesses.

INTRODUCTION

Many debilitating chronic illnesses are characterized by the presence of chronic fatigue (1). Indeed, chronic fatigue is the most commonly reported medical complaint of all patients seeking medical care (2). However, the fatigue syndromes, such as Chronic Fatigue Syndrome (CFS, sometimes called Myalgic Encephalomyelitis), Fibromyalgia Syndrome (FMS) and Gulf War Illnesses (GWI) are distinguishable as separate syndromes that have muscle and overall fatigue as major characteristics, among many other multiorgan signs and symptoms (3-6), including immune system abnormalities (7). These syndromes have complex chronic signs and symptoms, including muscle pain, chronic fatigue, headaches, memory loss, nausea, gastrointestinal problems, joint pain, vision problems, breathing problems, depression, low grade fevers, skin disorders, tissue swelling, chemical sensitivities, among others. Because of the complex nature of these illnesses, many patients are often diagnosed with multiple syndromes. Unfortunately, due to the lack of definitive laboratory or clinical tests that could identify the cause(s) of these illnesses, many patients are diagnosed with somatoforensic disorders. Often these patients have cognitive problems, such as short term memory loss, difficulty concentrating and psychological problems, that in the absence of contrary laboratory tests can result in practitioners diagnosing somatoform disorders rather than organic problems (6). Stress is often portrayed as an important factor in these disorders, and in fact stress can have many effects on the hormonal and immune systems that could be detrimental in virtually any chronic illness (8).

There is growing awareness that the chronic fatigue illnesses can have an infectious nature that is either responsible (causative) for the illness, a cofactor for the illness or appears as an opportunistic infection(s) responsible for aggravating patient morbidity (9). There are several reasons for this notion (10), including the nonrandom or clustered appearance of the illness, often in immediate family members, and the course of the illness and its response to therapies based on treatment of infectious agents. Since chronic illnesses are often complex, involving multiple, nonspecific, overlapping signs and symptoms, they are difficult to diagnose and even more difficult to treat (9). Most chronic fatigue illnesses do not have effective therapies, and these patients rarely recover from their condition (11), causing in some cases catastrophic economic problems.

SIGNS AND SYMPTOMS ANALYSIS

Some chronic illnesses, such as Rheumatoid Arthritis (RA), are well established in their clinical profile (12), whereas others, such as CFS, FMS and GWI, have rather nonspecific but similar overlapping, multi-organ signs and symptoms. A major difference between these illnesses appears to be in the severity of specific signs and symptoms. For example, CFS patients most often complain of chronic fatigue and joint pain, stiffness and soreness, whereas FMS patients have as their most major complaint muscle and overall pain, soreness and weakness. For the most part, the clinical profiles of these illnesses are similar, and this can be easily seen when the signs and symptoms of CFS, FMS, and GWI patients are compared (Figures 1A and IB). Thus although chronic illnesses are considered to be complex, they do display certain similarities, suggesting that these illnesses are related and not completely separate syndromes (6, 9). In addition, these chronic illness patients often show increased sensitivities to various environmental irritants and chemicals and enhanced allergic responses.

Although chronic fatigue illnesses have been known in the literature for many years, most patients with CFS, FMS, GWI and in some cases RA have had few treatment options. This may have been due to the imprecise nature of their diagnoses, which are usually based primarily on clinical observations rather than laboratory tests, and a lack of understanding about the underlying causes of these illnesses or the factors responsible for patient morbidity. Chronic illnesses could have different initial causes or triggers but similar secondary events, such as opportunistic viral and/or bacterial infections that cause significant morbidity (9, 10). With time these secondary events may progress to be the most important in determining overall signs and symptoms and treatment options.

The data presented in Figures 1A and IB show the most common signs and symptoms found in CFS, FMS and GWI patients and symptomatic GWI family members after the onset of illness. In these figures the data for FMS and CFS have been combined, because previous studies indicated that with the exception of the extent of muscle pain and tenderness, there were essentially no major differences in patient signs (6, 10). Illness Survey Forms were analyzed to determine the most common signs and symptoms at the time when blood was drawn from patients. The intensity of approximately 120 patient signs and symptoms prior to and after onset of illness was recorded on a 10-point rank scale (0-10, extreme). The data were arranged into 29 different signs and symptoms groups and were considered positive if the average value after onset of illness was two or more points higher than prior to the onset of illness. CFS/FMS patients had complex signs and symptoms that were similar to those reported for GWI, and the presence of rheumatoid signs and symptoms in each of these disorders indicates that there are also similarities to RA (12, 13). Moreover, it is not unusual to find immediate family members who slowly displayed similar signs and symptoms following the return home of veterans with GWI, suggesting that these civilian patients contracted their illnesses from chronically ill family members with GWI (10). Examination of the increase in signs and symptoms of GWI family members that now have a chronic illness similar to GWI indicates that they have signs and symptoms similar to civilian CFS/FMS patients. The main difference between veterans with GWI and their family members was in the greater breadth and severity of signs and symptoms found in GWI patients than in their symptomatic family members. Since Gulf War veterans were presumably exposed to many more environmental toxic agents compared to nondeployed family members, this is not unexpected. When the signs and symptoms of CFS/FMS/GWI were compared to
patients with other chronic illnesses that did not show evidence of chronic infections, there were also notable differences. For example, in contrast to CFS/FMS/GWI patients, this latter chronic illness patient group did not show differences in gastrointestinal problems, coagulation problems, hair loss and scalp problems, night sweats and intermittent fevers (Figures 1A and IB). This suggests that CFS/FMS/GWI patients with chronic infections may have some unique clinical problems not commonly found in other chronic illness patients.

CHRONIC INFECTIONS IN CFS, FMS AND GWI

As stated above, there exists indirect evidence suggesting the infectious nature in at least certain subsets of chronic illness patients. We have been particularly interested in the association of specific chronic infectious agents with CFS, FMS, GWI and RA, because these microorganisms can potentially cause most or essentially all of the signs and symptoms found in these patients (6, 9, 10, 13). One type of “stealth” infection that could fulfill the criteria of association with a wide range of signs and symptoms are certain microorganisms of the class Mollicutes. This is a class of small bacteria, lacking cell walls, and some species are capable of invading several types of human cells and tissues and are associated with a wide variety of human diseases (14).

We and others have examined the presence of mycoplasmal blood infections in CFS, FMS, GWI and RA patients. The clinical diagnosis of these disorders was obtained from referring physicians according to the patients’ major signs and symptoms. Blood was collected, shipped over night at 4°C and processed immediately for Nucleoprotein Gene Tracking (NPGT) after isolation of blood leukocyte nuclei (15, 16) or Forensic Polymerase Chain Reaction (FPCR) after purification of blood leukocyte DNA using a Chelex procedure (6, 13, 17). We used FPCR to determine the species of mycoplasmal infections. The sensitivity and specificity of the PCR methods were determined by examining serial dilutions of purified DNA of M. fermentans, M. pneumoniae, M. penetrans and M. hominis. Amounts as low as 10 fg of purified DNA were detectable. The amplification with genus primers produced the expected fragment size in all tested species, which was confirmed by hybridization with an inner probe 18). Others have also used PCR with single (19, 20) or multiple (21, 22) sets of PCR primers. Using NPGT to analyze the blood leukocytes from GWI patients we found that 91/200 (~45%) were positive for mycoplasmal infections. In contrast, in nondeployed, healthy adults the incidence of mycoplasmal infections was 4/62 (-6%) (15, 16, Table 1). Similarly, using PCR 55% of GWI patients were positive for Mycoplasma spp. and 36% were found to have M. fermentans infections (22, Table 1). The slight difference in percentage of positive patients is probably due to the differences in sensitivities of these two methods. In comparison, using FPCR or PCR 52-63% of CFS/FMS patients (n~l,000) had mycoplasmal infections (6, 19-23), whereas only 9-15% of controls (n~450) tested positive (Table 1).

Patients with CFS/FMS often have multiple mycoplasmal infections and probably other chronic infections as well. When we examined CFS/FMS patients for the presence of M. fermentans, M. pneumoniae, M. penetrans, M. hominis infections, multiple infections were found in over one-half of 93 patients (17, Table 1). CFS/FMS patients had double (>30%) or triple (>20%) mycoplasmal infections, but only when one of the species was M. fermentans or M. pneumoniae (17). We also found higher score values for increases in the severity of signs and symptoms in CFS/FMS patients with multiple infections. CFS/FMS patients with multiple mycoplasmal infections generally had a longer history of illness, suggesting that patients may have contracted additional infections during their illness (17).

CHRONIC INFECTIONS IN RA

The causes of rheumatic diseases are not known, but RA and other autoimmune diseases could be triggered or more likely exacerbated by infectious agents (24). In some animal species infection by certain species of mycoplasmas can result in remarkable clinical and pathological similarities to RA and other rheumatoid diseases. Aerobic and anaerobic intestinal bacteria, viruses and mycoplasmas have been proposed as important agents in RA (24-29), and there has been increasing evidence that mycoplasmas may play a role in the initiation or progression of RA (13, 29-31). Mycoplasmas have been proposed to interact nonspecifically with B-lymphocytes, resulting in modulation of immunity, autoimmune reactions and promotion of rheumatic diseases (30), and mycoplasmas have been found in the joint tissues of patients with rheumatic diseases, suggesting their pathogenic involvement (28).

When Haier et al. (13) and Vojdani and Franco (22) examined RA patients’ blood leukocytes for the presence of mycoplasmas, it was found that approximately one-half were infected with various species of mycoplasmas. The most common species found was M. fermentans, followed by M. pneumoniae and M. hominis and finally M. penetrans (13, 22). Similar to what we reported in CFS/FMS patients (17), there was a high percentage of multiple mycoplasmal infections in RA patients when one of the species was M. fermentans (13).

The precise role of mycoplasmas in RA and other rheumatic inflammatory diseases is under investigation; however, mycoplasmas could be important cofactors in the development of inflammatory responses in rheumatic diseases and for progression of RA. As an example of the possible role of mycoplasmas in rheumatic diseases, M. arthritidis infections in animals can trigger and exacerbate autoimmune arthritis (31, 32). This mycoplasma can also suppress T-cells and release substances that act on polymorphonuclear granulocytes, such as oxygen radicals, chemotactic factors and other substances (32). Mycoplasmal infections can increase proinflammatory cytokines, such as Interleukin-1, -2 and -6 (33), suggesting that they are involved in the development and possibly progression of rheumatic diseases such as RA.

A variety of microorganisms have been under investigation as cofactors or causative agents in rheumatic diseases (9, 24, 25). The discovery of EB virus (26) and cytomegalovirus (27) in the cells of the synovial lining in RA patients suggested their involvement in RA, possibly as a cofactor. There are a number of bacteria and viruses that are candidates in the induction or progression of RA or its progression (9, 24). In support of a bacterial involvement in RA, antibiotics like minocycline can alleviate the clinical signs and symptoms of RA (34). This and similar drugs are likely suppressing infections of sensitive microorganisms like mycoplasmas in rheumatic diseases, although they could also have immunoregulatory effects.

MYCOPLASMAL INFECTIONS IN OTHER DISEASES

Mycoplasmas have been associated with the progression of autoimmune and immunosuppressive diseases, such as FflV-AIDS (35). In some cases these infections have been associated with terminal human diseases, such as an acute fatal illness found with M. fermentans infections in non-AIDS patients (36). Importantly, mycoplasmal infections are now thought to be a major source of morbidity in FflV-AIDS (37). On this basis, Blanchard and Montagnier (37) have proposed that certain mycoplasmas like M. fermentans are important cofactors in the progression of FflV-AIDS, accelerating disease progression and accounting, in part, for the increased susceptibility of AIDS patients to additional opportunistic infections. Since most studies on the incidence of mycoplasmal infections in FflV-AIDS patients have employed relatively insensitive tests, it is likely that the occurrence of mycoplasmal infections in FflV-AIDS is much greater than previously thought and may be associated with a rapid fatal course of the disease. In FflV-AIDS mycoplasmas like M. fermentans can cause renal and CNS complications (38), and mycoplasmas have been found in various tissues, such as the respiratory epithelial cells of AIDS patients (39). Other species of mycoplasmas have been found in AIDS patients where they have also been associated with disease progression (40). In addition to immune suppression, some of this increased pathogenecity may be the result of mycoplasma-induced host cell membrane damage from toxic oxygenated products released from intracellular mycoplasmas (41). Also, mycoplasmas may regulate the FflV-1 virus, such as FflV-LTR-dependent gene expression (42), suggesting that mycoplasmas may play an important regulatory role in FflV expression.

There is some preliminary evidence that mycoplasmal infections are associated with various autoimmune diseases. In some mycoplasma-positive GWI cases the signs and symptoms of Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), Lupus, Graves’ Disease and other complex autoimmune diseases have been seen. Such usually rare autoimmune responses are consistent with certain chronic infections, such as mycoplasmal infections, that penetrate into nerve cells, synovial cells and other cell types. The autoimmune signs and symptoms could be the result of intracellular pathogens, such as mycoplasmas, escaping from cellular compartments and incorporating into their own structures pieces of host cell membranes that contain important host antigens that can trigger autoimmune responses. Alternatively, mycoplasma surface components, sometimes called ‘superantigens,’ may
directly stimulate autoimmune responses (43). Perhaps the most important event, the molecular mimicry of host antigens by mycoplasma surface components, may explain, in part, their ability to stimulate autoimmune responses (44).

Asthma, airway inflammation, chronic pneumonia and other respiratory diseases are known to be associated with mycoplasmal infections (45). For example, M. pneumoniae is a common cause of upper respiratory infections (46), and severe Asthma is frequently associated with mycoplasmal infections (47).

Cardiopathies can be caused by chronic infections, resulting in myocarditis, endocarditis, pericarditis and others. These are often due to chronic infections by Mycoplasma spp. (48), Chlamydia spp. (49) and possibly other infectious agents.

Mycoplasmal infections are also associated with a variety of illnesses, such as M. hominis infections in patients with hypogammaglobulinemia (29), and M. genitalium with nongonococcal urethritis (50). Mycoplasmas can exist in the oral cavity and gut as normal flora, but when they penetrate into the blood and tissues, they may be able to cause or promote a variety of acute or chronic illnesses. These cell-penetrating species, such as M. penetrans, M. fermentans, M. hominis and M. pirum, among others, can cause infections that result in complex systemic signs and symptoms. Mycoplasmal infections can also cause synergism with other infectious agents. Similar types of chronic infections caused by Chlamydia, Brucella, Coxiella or Borriela may also be present either as single agents or as complex, multiple infections in many chronic illnesses (9).

MYCOPLASMA TREATMENT

Although mycoplasmal infections are often misdiagnosed or inappropriately treated (45), they can be successfully treated using antibiotics and nutritional support (51, 52). Appropriate treatment with antibiotics should result in patient improvement and even recovery, and this has been seen in GWI, CFS, FMS and RA patients (Table 2). The recommended treatments for mycoplasmal blood infections require long-term antibiotic therapy, usually 12 months or more or multiple 6-week cycles of doxycycline (200-300 mg/day), ciprofloxacin (1,500 mg/day), azithromycin (500 mg/day) or clarithromycin (750-1,000 mg/day). Multiple cycles are required, because only a few patients recovered after a few cycles, possibly because of the intracellular locations of pathogenic mycoplasmas, the slow-growing nature of these microorganisms and their relative drug sensitivities. For example, of 87 GWI patients that tested positive for mycoplasmal infections, all patients relapsed after the first 6-week cycle of antibiotic therapy, but after up to 6-7 cycles of therapy 69/87 patients responded and eventually recovered and returned to active duty (15, 16, Table 2). Similarly, the majority of CFS/FMS patients who tested positive for mycoplasmal infections also responded to the antibiotic therapy (53, Table 2). Although these clinical studies were not placebo-controlled, blinded studies, double-blind, placebo-controlled antibiotic trials using minocycline versus placebo treatment of RA patients indicates that this antibiotic is clinically effective in RA (34, 54, Table 2).

The clinical responses that were seen in mycoplasma-positive chronic illness patients were not due to placebo effects, because administration of some antibiotics, such as penicillins, resulted in patients becoming more not less symptomatic, and they were not due to immunosuppressive effects that can occur with some of the recommended antibiotics (6, 9, 16). Interestingly, CFS, FMS and GWI patients that slowly recover after several cycles of antibiotics are generally less environmentally sensitive, suggesting that their immune systems may be returning to pre-illness states. If these illnesses were caused by psychological problems or solely by environmental exposures rather than infections, they should not respond to the recommended antibiotics and slowly recover. In addition, if such treatments were just reducing autoimmune responses, then patients should relapse after the treatments are discontinued, and this is not what has been found. CFS, FMS, RA or GWI patients also have nutritional and vitamin deficiencies that must be corrected (52, 53). In addition, a fully functional immune system may be essential to overcoming these infections, and supplements and immune enhancers appear to be effective in helping patients recover (52, 53).

Although we have proposed that chronic infections are an appropriate explanation for the morbidity seen in a rather large subset of CFS, FMS, GWI and RA patients, and in a variety of other chronic illnesses, not every patient will have this as a diagnostic explanation or have the same types of chronic infections. Additional research will be necessary to clarify the role of multiple infections in chronic diseases, but these patients could benefit from appropriate antibiotic and neutraceutical therapies that alleviate morbidity.

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26. Fox, R.I., Luppi, M., Pisa, P. et al. Potential role of Epstein-Bar virus in Sjogren’s syndrome and rheumatoid arthritis. Journal of Rheumatology 1992; 32(Suppl): 18-24.
28. Schaeverbeke, T., Renaudin, H. Clerc, M. et al. Systematic detection of mycoplasmas by culture and polymerase chain reaction (PCR) procedures in 209 synovial fluid samples. Reviews Rheumatology 1997; 64: 120-128.
29. Furr, P.M., Taylor-Robinson, D. and Webster, A.D.B. Mycoplasmas and ureaplasmas in patients with hypogammaglobulinemia and their roll in arthritis: microbiological observation over twenty years. Annuals of Rheumatological Diseases 1994; 53: 183-187.
30. Simecka, J.W., Ross, S.E., Cassell, G.H. and Davis, J.K. Interactions of mycoplasmas with B cells: production of antibodies and nonspecific effects. Clinical Infectious Diseases 1993; 17 (Supp. 1): S176-S182.
31. Cole, B.C. and Griffith, M.M. Triggering and exacerbation of auroimmune arthritis by the Mycoplasma arthritidis superantigen MAM. Arthritis and Rheumatology 1993;36:994-1002.
32. Kirchhoff, H., Binder, A., Runge, M. et al. Pathogenic mechanisms in the Mycoplasma arthritidis polyarthritis of rats. Rheumatology Int. 1989; 9: 193-196.
33. Muhlradt, P.F., Quentmeier, H. and Schmitt, E. Involvement of interleukin-1 (IL-1), IL-6, IL-2 and IL-4 in generation of cytolytic T cells from thymocytes stimulated by a Mycoplasma fermentans-derived product. Infection and Immunology 1991; 58: 1273-1280.
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36. Lo, S.-C, Dawson, M.S., Newton, P.B. et al. Association of the virus-like infectious agent originally reported in patients with AIDS with acute fatal disease in previously healthy non-AIDS patients. American Journal of Tropical Medicine and Hygiene 1989; 41: 364-376.
37. Blanchard, A. and Montagnier, L. AIDS associated mycoplasmas. Annual Review of Microbiology 1994; 48: 687-712.
38. Bauer, FA., Wear, D.J., Angritt, P. and Lo, S.-C. Mycoplasmal fermentans (incognitus strain) infection in the kidneys of patients with acquired immunodeficiency syndrome and associated nephropathy: a light microscopic, immunohistochemical and ultrastructural study. Human Pathology 1991; 22, 63-69.
39. Sloot, N, Hollandt, H. Gatermann, S. and Dalhoff, K. Detection of Mycoplasma spp. in bronchoalveolar lavage of AIDS patients with pulmonary infiltrates. Zentralbl Bacteriology 1996; 284: 75-79.
40. Grau, O., Slizewicz, B. Tuppin, P. et al. Association of Mycoplasma penetrans with human immunodeficiency virus infection. Journal of Infectious Disease 1995;172:672-681.
41. Pollack, J. D., Jones, M. A. and Williams, M. V. The metabolism of ADIS-associated mycoplasmas. Clinical Infectious Diseases 1993; 17: S267-S271.
42. Nir-Paz, R., Israel, S., Honigman, A. and Kahane, I. Mycoplasmas regulate HIV-LTR-dependent gene expression. FEMS Microbiology Letters 1995; 128: 63-68.
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44. Baseman, J.B., Reddy, S.P. and Dallo, S.F. Interplay between mycoplasma surface proteins, airway cells and proetin manifestations of mycoplasma-mediated human infections. American Journal of Respiratory Critical Care Medicine 1996;154: S137-S144.
45. Cassell, G.H. Infectious causes of chronic inflammatory diseases anticancer. Emerging Infectious Diseases 1998;4:475-487.
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47. Gil, J.C., Cedillo, R.L., Mayagoitia, B.G and Paz, M.D. Isolation of Mycoplasma pneumoniae from asthmatic patients. Annuals of Allergy 1993; 70: 23-25.
48. Prattichizzo, F.A., Simonetti, I. and Galetta, F. Carditis associated with Mycoplasma pneumoniae infections. Clinical aspects and therapeutic problems. Minerva Cardioangiology 1997; 45: 447-450.
49. Fairley, C.K.I, Ryan, M, Wall, P.G. and Weinberg, J. The organisms reported to cause infective myocarditis and pericarditis in England and Wales. Journal of Infection 1996; 32: 223-225.
50. Busolo, F., Camposampiero, D, Bordignon, G. and Bertollo, G.
Detection of Mycoplasma genitalium and Chlamydia trachomatis DNAs in male patients with urethritis using the polymerase chain reaction. New Microbiologia 1997; 20: 325-332.
51. Nicolson, G.L. and Nicolson, N.L. Doxycycline treatment and Desert Storm. JAMA 1996; 273: 618-619.
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53. Nicolson, G.L. The role of microorganism infections in chronic illnesses: support for antibiotic regimens. CFIDS Chronicle 1999; 12(3): 19-21.
54. O’Dell, J.R., Paulsen, G. Haire, C.E. et al. Treatment of early seropositive rheumatoid arthritis with minocycline: four-year follow-up of a double-blind, placebo-controlled trial. Arthritis and Rheumatology 1999; 42: 1691-1695.

FIGURE LEGENDS

Figures 1A and IB (same legend). Incidence of increase in severity of signs and symptoms in 260 chronic illness patients. Severity of illness was scored using 117 signs and symptoms on a 10-point scale (0, none; 10 extreme) prior to and after the onset of illness. Scores were placed into 29 categories containing 3-9 signs/symptoms and were recorded as the sum of differences between values before and after onset of illness divided by the number of questions in the category. Changes in score values of 2 or more points were considered relevant. Patient groups were CFS/FMS (■), GWI (G) GWI symptomatic family members (G) and chronic illness patients not in the above groups that did not show evidence of chronic bacterial infection ( G). Asterisk (*) indicates score = 0.
Table 1. Summary of mycoplasmal infections in patient groups and controls.

Jörg Haier 1, Marwan Nasralla 1, A. Robert Franco 2, and Garth L. Nicolson 3
1 The Institute for Molecular Medicine, 16371 Gothard St. H, Huntington Beach, CA 92647
2 The Arthritis Center of Riverside, Riverside, CA 92501

SUMMARY

Objectives: Mycoplasmal infections are associated with several acute and chronic illnesses. Some mycoplasmas can enter a variety of tissues and cells and cause system-wide or systemic signs and symptoms. 


Methods: Patients (14 female, 14 male) diagnosed with Rheumatoid Arthritis (RA) were investigated for mycoplasmal infections in their blood leukocytes using a forensic Polymerase Chain Reaction (PCR) procedure. Amplification was performed with genus- and species-specific primers, and a specific radio-labeled internal probe was used for Southern hybridization with the PCR product. Patients were investigated for presence of Mycoplasma spp., and positive cases were further tested for infections with the following species: M. fermentans, M. hominis, M. pneumoniae and M. penetrans. 


Results: The Mycoplasma spp. sequence, which is not entirely specific for mycoplasmas, was amplified from the peripheral blood of 15/28 patients (53.6 %), and specific PCR products could not be detected in 13 patients (46.4 %). Significant differences (p<0.001) were found between patients and positive healthy controls in the genus-test (3/32) and in the specific tests (0/32). Moreover, the incidence of mycoplasmal infections was similar in female and male patients. Using species-specific primers, we were able to detect infections of M. fermentans (8/28), M. pneumoniae (5/28), M. hominis (6/28) and M. penetrans (1/28) in RA patients. In 36% of the patients we observed more than one mycoplasma species in the blood leukocytes. All multiple infections occurred as combinations of M. fermentans with other species.

Conclusions: The results suggest that a high percentage of RA patients have systemic mycoplasmal infections. Systemic mycoplasmal infections may be an important cofactor in the pathogenesis of RA, and their role needs to be further explored.

Introduction

Mycoplasmas are the smallest self-replicating, pleotrophic bacteria that lack cell walls [1,2]. The largest group of the class Mollicutes is divided into more than 100 mycoplasma species, which are further subclassified into various strains. Mycoplasmas are often found as extracellular parasites attached to the external surfaces of host cells, but some species invade host tissues and cells, and replicate intracellularly. These microorganisms can produce a variety of effects on host cells and tissues. Besides affecting cell growth and morphology, mycoplasmas are able to alter metabolic, immunological and biochemical functions [3].

Mycoplasmas are commonly found in the oral cavity and as symbiotic gut flora. Formerly, mycoplasmas were considered as relative benign microorganisms with a low pathogenic potential. When they penetrate into blood vessels and colonize major organs, certain species can, however, cause acute and chronic illnesses. Some mycoplasmas, such as M. penetrans, M. fermentans and M. pirum, can enter a variety of tissues and cells and cause a broad spectrum of signs and symptoms [3]. Mycoplasmas have also been shown to have a complex relationship with the immune system [4]. They are very effective at evading host immune responses, and synergism with other infectious agents has been seen. The best known species is M. pneumoniae, which can cause atypical pneumonia [5,6]. Mycoplasmal infections can present as different clinical disorders with acute and chronic signs and symptoms. Although many of these signs and symptoms are nonspecific, they seem to be related, in part, to immunological or autoimmunological responses. For example, using culturing techniques Ureaplasma urealyticum, M. pneumoniae and M. salivarium have been localized in the joint tissues of patients with rheumatoid diseases [7]. Hoffman et al. [8] found serological evidence for active and inactive mycoplasmal infections in patients with rheumatoid arthritis (RA) and juvenile RA, but they could not detect mycoplasmal DNA in the synovial fluid of these patients using polymerase chain reaction (PCR). Other studies observed immunological evidence for mycoplasmal infections in RA patients [9,10].

We have begun to examine patients with chronic illnesses for the presence of systemic mycoplasmal infections. In recent studies we have shown that patients with Chronic Fatigue Syndrome (CFS) and/or Fibromyalgia Syndrome (FMS) have a much higher incidence of mycoplasmal infections in their blood leukocytes than healthy controls without clinical signs and symptoms [11-13]. We hypothesized that chronic mycoplasmal infections might be also related to the pathogenesis of other chronic illnesses, such as RA.

Mycoplasmal infections are usually diagnosed by serological procedures or culture techniques [14,15]. Both of these techniques are very limited in their sensitivity, and thus mycoplasmal infections are often underdiagnosed or misdiagnosed [16]. The introduction of mycoplasma-specific primers in PCR enables sensitive and specific detection of mycoplasmal infections and discrimination between different mycoplasma species. Using PCR techniques the presence of mycoplasmas was investigated in synovial fluids of patients with RA and other chronic arthritides. Schaeverbeke et al. [17] showed that M. fermentans, but not M. penetrans was detectable in 20% of these patients and other types of arthritis of unknown causes agent, but not in patients with reactive, posttraumatic or chronic juvenile arthritis. Additionally, M. genitalium was found in some RA patients [18]; however, the sensitivity of the conventional PCR procedures was not satisfying [19]. The forensic PCR method that we use to identify mycoplasmal infections is very sensitive and highly specific [11].

In this preliminary study, we report on the detection of mycoplasmas in blood leukocytes of patients with RA. Using a sensitive forensic PCR method with genus-specific primers, we investigated blood samples for the presence of any type of mycoplasmal infection. Using species-specific primers, we then tested for the presence of several mycoplasma species.

Materials and Methods

Patients
Blood samples from 28 patients (50% female, 50% male), diagnosed with RA were investigated for mycoplasmal infections in their blood leukocytes. According to the American College of Rheumatology modified criteria were used for patient’s diagnosis [20]. All patients were examined by a rheumatologist (A.R.F.) and all patients fulfilled the ACR classification criteria for RA. Patients’ age ranged between 22 and 65 years (median 42 years). The duration of RA history was 16 to 300 months (median=149 months). All patients had no antibiotic treatments for at least 6 weeks before the blood was drawn.

Specimens
Specimens were collected and treated as previously described [11]. Briefly, blood was collected in citrate-containing tubes and immediately brought to ice bath temperature. Samples were shipped refrigerated or on wet ice by over night courier. Whole blood (50 l) or blood leukocytes were used for preparation of DNA using Chelex (Biorad) as follows. Blood cells were lysed with nanopure water (1.3 ml) at room temperature for 30 min. After centrifugation at 13,000 x g for 2 min, the supernatants were discarded. Chelex solution (200 l) was added, and the samples were incubated at 56C and at 100C for 15 min each. Aliquots from the centrifuged samples were used immediately for PCR or stored at -70C until use.

Amplification
Amplification of the target sequences (Table 1) was performed in a total volume of 50 l PCR buffer (10 mM Tris-HCl, 50 mM KCl, pH 9) containing 0.1% Triton X-100, 200 m each of dATP, dTTP, dGTP, dCTP, 100 pmol of each primer, and 0.5-1 g of chromosomal DNA. Purified Mycoplasmal DNA (0.5-1ng of DNA) was used as a positive control for amplification. The amplification was carried out for 40 cycles with denaturing at 94C. Annealing was performed at 60C (genus-specific primers and M. penetrans) or 55C (M. pneumoniae, M. hominis and M. fermentans). Extension temperature was 72C in all cases. Finally, product extension was allowed at 72C for 10 min. [11,21-23]. Negative and positive controls were used in each experimental run.

Southern Blot Confirmation
The amplified samples were run on a 1% agarose gel containing 5 l/100 ml of ethidium bromide in TAE buffer (0.04 M Tris-Acetate, 0.001 M EDTA, pH 8.0). After denaturing and neutralization, Southern blotting was performed as follows. The PCR product was transferred to a Nytran membrane. After transfer, UV cross-linking was performed. Membranes were prehybridized with hybridization buffer consisting of Denhardt’s solution and 1 mg/ml salmon sperm as blocking reagent. Membranes were then hybridized with 32P-labeled corresponding internal probe (107 cpm per bag) (see Table 1). After hybridization and washing to remove unbounded probe, the membranes were exposed to autoradiography film for 7 days at -70C.

Results
For the detection of mycoplasmal infections in blood leukocytes, we first used genus-specific primers. The Mycoplasma spp. sequence was amplified from DNA extracted from the peripheral blood of 15/28 (53.6 %) patients, whereas specific PCR products were not detected in the 13 negative patients (46.4 %). Results were similar in female and male patients. In 32 healthy controls without any clinical signs and symptoms, positive results were shown in 3 cases (9.4%) for Mycoplasma spp. test but not for the other species-specific tests (0/32).

Specific primers for M. fermentans, M. pneumoniae, M. penetrans and M. hominis were used to detect species-specific mycoplasmal DNA by PCR. In 10/15 patients with a positive signal for M. spp. we detected one or more mycoplasma species, but in 5 positive patients we were unable to find at least one of the four tested species. The incidence of infections with M. fermentans (8/28), M. pneumoniae (5/28) and M. hominis (6/28) was similar. M. penetrans was found in only one patient. In 36% of the patients that tested positive for the general mycoplasmal infection, we observed more than one species in the blood leukocytes. These multiple infections occurred as combinations of M. fermentans with other species. Single infections were found in 5 patients (M. fermentans n=2; M. hominis n=2; M. pneumoniae n=1), but were not observed with M. penetrans. All four species were detected in one patient.

Although the GPO-1 and UNI-sequences are capable of a few possible cross-reactions with mycoplasma-related organisms, the conditions used yielded specific products for mycoplasmas as shown by van Kuppeveld et al. [24] and Dussurget et al. [25]. That the patients we examined had mycoplasmal infections was confirmed by species analysis using PCR. Using the M. fermentans-specific primers SB1 and SB2 from the tuf gene we found a single band of 850 bp size that hybridized only with the 32P-labeled internal probe SB3. Similar results were obtained for the other mycoplasma species (see figure 1). To examine the reliability of the method we performed multiple assays (repeated 3-7 times) on 40 samples with other diagnoses. All results were completely reproducible. In three cases, the sixth and seventh repeat of an initial positive result produced only a week but positive signal due to degradation of DNA.

Fresh blood and immediate DNA preparation resulted in better results than blood that was processed after a period of time at room temperature. Six positive blood samples were divided into 5 aliquots each and stored at room temperature for different time intervals (processed immediately or after 1, 2, 4, or 7 days). Over time the PCR signal decreased. In all samples that showed positive results in fresh DNA preparations, the PCR signal became weak after 2 days of blood storage at room temperature. After 4 days, negative results were obtained in 4 cases, whereas the other two samples showed very faint bands. No specific PCR product was detectable after one week. Additionally, blood collected in tubes containing citrate gave better results than blood collected in acid-EDTA.

The sensitivity of mycoplasma detection by the described method was assessed by the detection of control mycoplasma DNA and by internal Southern hybridization using mycoplasma-specific probes. Using serial dilutions of mycoplasma DNA, the method was able to detect as low as 1 fg of DNA [11]. In other experiments, M. fermentans was added to control blood samples at various concentrations. We were able to detect specific products down to 10 ccu/ml blood. Thus with the use of specific Southern hybridization this PCR procedure can result in specific test results of high sensitivity, down to the presence of approximately a single microorganism in a clinical sample.

In our experience, conventional PCR yields similar results to forensic PCR with extracellular mycoplasma, but not with clinical samples that contain intracellular mycoplasmas. The reason for this is not known, but it could be due to inhibitors present in the clinical samples or to loss of mycoplasma DNA in the conventional extraction procedures due to protein complexing.

Discussion

Although the underlying causes of RA are not known, RA and other autoimmune diseases could be triggered, at least in part, by infectious agents. The remarkable clinical and pathological similarities between certain infectious diseases in animal species and those of some human rheumatic illnesses, such as RA, have encouraged the search for a microbial etiology for these syndromes. A long list of microorganisms, including aerobic and anaerobic intestinal bacteria, several viruses and mycoplasmas have been proposed as important in these illnesses [26]. Although several initial findings on many etiological agents were corroborated by further studies, the concept of a microbial trigger for RA is attractive. Recently, there has been increasing evidence that mycoplasmas may, in part, play a role in the genesis of arthritis [27].

In the present pilot study we detected several mycoplasma species in blood leukocytes of patients suffering from RA. Although the patient numbers in these studies were not large, using a highly sensitive and specific PCR technique we were able to detect mycoplasmal DNA in more than 50% of patients. Mostly we detected M. fermentans, and M. penetrans was found in only one patient with multiple mycoplasmal infections. Recently, similar findings were published using synovial fluids and joint tissue specimens [17]. Additionally, we observed infections with M. pneumoniae and M. hominis. The presence of trace amounts of mycoplasmal antigens for these species or specific antibodies against mycoplasma species were found in other studies using immunological methods [10, 14]. Interestingly, we detected multiple infections with several mycoplasma species in a high percentage of our patients, but these multiple infections were seen only in combination with M. fermentans infections. The UNI- and GPO1 primer are not totally genus-specific. However, the conditions used for PCR yield amplification products with a high degree of specificity and sensitivity [24, 25]. To overcome the problems regarding the limited specificity we confirmed the results for the Mycoplasma spp. assay with highly species-specific assays. We were able to identify at least one mycoplasma species in 10 of 15 patients where the general test was positive. In the remaining 5 patients it is more likely that other mycoplasma species were responsible for the positive amplification signal, such as M. arthritidis, rather than cross-reactions with other closely related microorganisms. However, the limited specificity of the general test cannot completely rule out such cross-reactivity. Future studies will include more mycoplasmal species using highly species-specific primers.

Since little is known about the possible involvement of mycoplasmas in the pathogenesis of chronic diseases, it remains uncertain whether our findings represent a causal agent, cofactor, or secondary superinfection in patients with immundisturbances. However, mycoplasmas are able to induce immundysfunctions and autoimmune reactions. Thus, mycoplasmal infections may be, in part, involved in the pathogenesis of RA.

Mycoplasmal infections were reported in patients with various inflammatory diseases, such as endocarditis [28], pericarditis [29] or encephalomyelitis [30], where immunological or autoimmunological phenomena coexist. Although the basis for these infections is not well understood, it is apparent that several species of pathogenic mycoplasmas are endowed with a sophisticated genetic machinery for altering their surface attributes. This surface phenotypic variation is thought to play a key role in the establishment and persistence of mycoplasma infections by enabling evasion of host defenses and by ensuring adaptation to the rapidly changing microenvironmental conditions encountered in the host [31]. Nonspecific interactions between mycoplasmas and B-lymphocytes have been implicated in disease pathogenesis, possibly leading to autoimmune reactions, modulation of immunity, and/or promotion of lesion development [32]. The potential role of mycoplasmas in various joint diseases remains unknown but they could be an important factor or cofactor. Thus the complex relationship between mycoplasmal infections and the immune system of the host may be, in part, responsible for the pathogenesis of rheumatological inflammatory diseases. For example, M. arthritidis-related superantigens were found to compromise T-cells [33], and they can trigger and exacerbate autoimmune arthritis in animal models. Furthermore, this mycoplasma species releases substances that act on polymorphnuclear granulocytes, such as oxygen radicals, and chemotactic and aggregating substances [34]. Several studies have shown that mycoplasmal infections lead to increased levels of proinflammatory cytokines, such Interleukin-1, -2, -4 and –6 [35,36]. Therefore, M. arthritidis and possibly other species may be responsible, in part, for autoimmune phenomena at the early stages of RA, and in their progression. Deficient or aberrant immune responses (or other underlying diseases) might be necessary for the development and progression of RA and other rheumatological illnesses.

Other microorganisms are still under investigation as causative agents or important cofactors for these chronic diseases. Reports about the detection of Epstein-Barr virus or cytomegalovirus in synovial specimen are controversial [38-40]. Furthermore, retroviruses and enteropathogenic bacteria continue to be intensively discussed as possible etiologic factors of RA [40,41]. The identification of mycoplasmal infections in the leukocyte blood fractions of a rather large subset of RA patients support the hypothesis that mycoplasmas, and probably other chronic infections as well, may be an important source or cofactor for morbidity in these patients. Further investigation of the potential role of mycoplasma in RA patients will require comparison with other forms of arthritis and chronic inflammatory diseases.

Recently, it was found that minocycline is an interesting new drug for the treatment of RA. Tetracycline compounds have long been used by rheumatologists, and their antirheumatic activity has been demonstrated [42,43]. The reason why minocycline alleviates the clinical signs and symptoms of RA is unclear, but the responses of some patients with RA to minocycline might be due to the susceptibility of mycoplasmas to tetracyclines.

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Table 1.
Sequences from mycoplasmal DNA used for mycoplasma genus -specific and species-specific PCR. Specificity of each primer was evaluated using Blast-Search program on the GenBank.