Garth L. Nicolson,(1) PhD, Marwan Y. Nasralla,2 PhD, Nancy L. Nicolson,(1) PhD, Joerg Haier,(3) MD, PhD (Journal of Chronic Fatigue Syndrome 2002)
(1)The Institute for Molecular Medicine, Huntington Beach, California, USA, (2)International Molecular Diagnostics, Inc., Huntington Beach, California, USA, (3)Department of Surgery, University Hospital Muenster, Germany
Correspondence: Prof. Garth L. Nicolson, Office of the President, The Institute for Molecular Medicine, 15162 Triton Lane, Huntington Beach, California 92649. Tel: 714-903-2900; Fax: 714-379-2082; Website: www.immed.org; Email: firstname.lastname@example.org
Immediate family members of veterans diagnosed with Gulf War Illnesses (GWI) often complain of fatiguing illnesses, and upon analysis they report similar signs and symptoms as their veteran family members. Since a relatively common finding in GWI patients is a bacterial infection due to Mycoplasma species, we examined military families (149 patients: 42 veterans, 40 spouses, 32 other relatives and 35 children with at least one family complaint of illness) selected from a group of 110 veterans with GWI who tested positive (~41%) for at least one of four Mycoplasma species: M. fermentans, M. hominis, M. pneumoniae or M. genitalium. Consistent with previous results, over 80% of GWI patients who were positive for blood mycoplasmal infections had only one Mycoplasma species, M. fermentans.
In healthy control subjects, the incidence of mycoplasma1 infection was ~8.5% and none were found to have multiple mycoplasmal species (P<0.001). In 107 family members of Mycoplasma-positive GWI patients there were 57 patients (53%) that had essentially the same signs and symptoms as the veterans and were diagnosed with Chronic Fatigue Syndrome (CFS) and/or Fibromyalgia Syndrome. Most of these CFS patients also had mycoplasmal infections compared to the few non-symptomatic family members (P<0.001), and the most common species found was M. fermentans. In contrast, in the few non-symptomatic family members that tested mycoplasma-positive, the Mycoplasma species were often different from the species found in the GWI patients.
The results suggest that a subset of GWI patients have mycoplasmal infections, possibly obtained as contaminants from multiple vaccines given during deployment, and these infections can be transmitted to immediate family members who subsequently display similar signs and symptoms and are diagnosed with CFS and/or Fibromyalgia Syndrome.
KEYWORDS. Gulf War Syndrome, bacterial infections, Chronic Fatigue Syndrome, Myalgic Encephalomyelitis, Fibromyalgia Syndrome, infectious disease
A sizable fraction (estimated at over 16%) of the veterans of the 1991 Persian Gulf War from the United States, Great Britain, Canada, Australia and other nations slowly developed, usually with a lag of 6-18 months or more after the conflict, a variety of complex chronic signs and symptoms. These were characterized by disabling fatigue, intermittent low-grade fevers, night sweats, arthralgias, myalgias, short-term memory loss, confusion, irritability, depression, headaches, skin rashes, intermittent diarrhea, respiratory complaints, photophobia and other signs and symptoms (1-4). These illnesses have been called Gulf War Syndrome or Gulf War Illnesses (GWI) (5).
Since routine laboratory test results on GWI patients are usually not consistent with a single, specific diagnosis, veterans often did not receive a diagnosis for their condition (illness of unknown origin), or they received a diagnosis of somatoform disorder (1). The signs and symptoms of GWI do not easily fit into ICD-10 diagnostic categories, but they loosely fall into the category of fatiguing illnesses (2-4), and the signs and symptoms of GWI resemble Chronic Fatigue Syndrome or Myalgic Encephalomyelitis (CSF/ME) (2, 6-9).
We (6-9) found that most of the signs and symptoms in a large subset of GWI patients could be explained by chronic pathogenic bacterial infections. Evidence for infectious agents has been found in GWI patients’ urine (11) and blood (6-10). In studies on hundreds of U.S. and British veterans with GWI, approximately 40-50% of GWI patients show evidence of mycoplasmal infections compared to 6-9% in non-deployed, healthy subjects (6-10). This has also been confirmed in a large study of 1,600 veterans at over 30 veterans’ and defense medical centers (VA Cooperative Clinical Study Program #475) in the United States (12).
Mycoplasmas are small intracellular bacteria lacking cell walls and extensive intracellular organells. Historically, mycoplasmal infections were thought to produce relatively mild diseases limited to particular tissues or organs, such as urinary tract or respiratory infections. However, the Mycoplasma species detected in GWI patients with molecular techniques are highly virulent, colonize a wide variety of organs and tissues, and are difficult to treat (13-15). The mycoplasma most commonly detected in GWI, Mycoplasma fermentans, was found in the overwhelming majority of those GWI patients positive for any mycoplasma (7-10).
M. fermentans is an intracellular Mycoplasma, and it is unlikely that this type of infection will result in a strong antibody response. This may explain the lack of a high prevalence of serologic evidence for these types of intracellular infections in GWI patients compared to nondeployed controls (15).
When examining GWI patients for illness, we noticed that immediate family members often had similar complaints (9, 16). Previously a U.S. Congressional committee had questioned approximately 1,200 families where one member was a veteran with GWI and found that 77% of spouses and 65% of children born after the Gulf War had similar health complaints as the veteran (17). This suggested that in these families GWI was being transmitted from veterans with GWI to immediate family members. The most likely type of transmitted agent would be the chronic infections found in Gulf War veterans, such as mycoplasmal infections (6-10).
Therefore, we examined the family members of Gulf War veterans who were positive for mycoplasmal infections to see if their signs and symptoms and chronic infections were similar. We found that most family members of GWI patients that were symptomatic with signs and symptoms of CFS/ME also had mycoplasmal infections.
MATERIALS AND METHODS
Gulf War veterans with GWI and a positive test for mycoplasmal infection and their immediate family members (149 patients: 42 veterans, 40 spouses, 32 other relatives and 35 children) were enrolled (Table 1). Seventy age-matched healthy volunteers were recruited and used as control subjects.
All subjects underwent a medical history and routine laboratory tests. If necessary, medical records were also reviewed to determine if patients suffered from organic or psychiatric illnesses that could explain their symptoms (3).
When positive results were found in any of the evaluations of civilians that met the Fukuda et al. (18) exclusionary criteria for CFS, the patients were not included in the study. All subjects completed an illness survey questionnaire (Illness Survey Form, www.immed.org/signsympt.htm), which included demographic information, known environmental exposures, dates of illness onset, health status before and immediately after the Gulf War and current health status. Additionally, all subjects were questioned about medication use during the three months prior to the study, and they had to be free of antibiotic treatment for three months prior to blood collection. Controls had to be free of disease for at least three months prior to data collection.
Blood was collected in EDTA-containing tubes and immediately brought to ice bath temperature as described previously (19, 20). Samples were shipped with wet ice by air courier to the Institute for Molecular Medicine and International Molecular Diagnostics, Inc. for analysis. All blood samples were blinded. Whole blood (50 µl) was used for preparation of DNA using Chelex (Biorad, Hercules, USA) as follows. Blood cells were lysed with nano-pure 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 ml) was added, and the samples were incubated at 56°C and at 100°C for 15 minutes each. Aliquots from the centrifuged samples were used immediately for PCR or stored at -70°C until use. Multiple Mycoplasma tests were performed on all patients (19, 20).
Amplification of Gene Sequences
Amplification of the target gene sequences (Table in Nasralla et al., ref. 19) containing 0.1% Triton X-100, 200 mm each of dATP, dTTP, dGTP, dCTP, 100 pmol of each primer, and 0.5 – 1 mg of chromosomal DNA. Purified mycoplasmal DNA (0.1 – 1 ng of DNA) was used as a positive control for amplification. The amplification was carried out for 40 cycles with denaturing at 94°C and annealing at 60°C (genus-specific primers and M. penetrans) or 55°C (M. pneumoniae, M. hominis, M. fermentans, M. genitalium). Extension temperature was 72°C in all cases. Finally, product extension was performed at 72°C for 10 min. Negative and positive controls were present in each experimental run (19, 20).
Southern Blot Confirmation
The amplified samples were run on a 1% agarose gel containing 5 µl/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 1x Denhardt’s solution and 1 mg/ml salmon sperm DNA as blocking reagent. Membranes were then hybridized with 32P-labeled internal probe (107 cpm per bag). After hybrization and washing to remove unbounded probe, the membranes were exposed to autoradiography film for 1- 2 days at -70°C (19, 20).
Subjects’ demographic characteristics were assessed using descriptive statistics and students’ t-tests (independent samples test, t-test for equality of means, 2-tailed). The 95% confidence interval was chosen. Pearson Chi-Square test was performed to compare prevalence data between patients and control subjects. Additionally, sex differences were investigated using Pearson Chi-Square test. Illness survey data were statistically analyzed using Spearman Rank correlation and Mann-Whitney tests.
As found in previous studies (9,16), veterans of the Gulf War with chronic illnesses (GWI) exhibited multiple signs and symptoms (Figures 1A, B). Upon examination, the signs and symptoms of GWI were indistinguishable from civilian patients diagnosed with CFS/ME (Figures 1A, B c.f. ref. 9).
Examination of the signs and symptoms of approximately equal numbers of male and female GWI patients indicated that there were no significant differences between their general signs and symptoms (data not shown). When family members who were symptomatic with chronic signs and symptoms and diagnosed with CFS/ME were examined (Table 1, age range 4-49 years), they also appeared to have signs and symptoms indistinguishable from veterans with GWI (Figures 1A, B). When CFS-symptomatic GWI family members were grouped into male/female or adults/children, there were no significant differences between the signs and symptoms found in these groups. The only signs/symptoms differences found were in symptomatic family members who did not test positive for mycoplasmal infections (Figures 1A, B).
Similar to previous studies (6-10) 45 of 110 GWI patients or ~41% had mycoplasmal infections (Table 2), and almost all of these (37 out of 45 or ~82%) were single infections (one species of mycoplasma) (Table 3). M. fermentans was found in ~85% of these single infection cases. When the few multiple infection cases were examined, most were found to have combinations of M. fermentans plus either M. pneumoniae or M. genitalium (Table 3). In contrast, in healthy control subjects only 6 of 70 subjects (8.5%) were positive for mycoplasmal infections, and all of these were single species infections of various types (Table 4).
Comparing GWI patients and non-symptomatic control subjects, there was a significant difference in the incidence of mycoplasmal infections (P<0.001). However, significant differences in infection incidence or species of mycoplasmal infection between male and female GWI patients or control subjects were not seen in these patient groups.
In family members of Gulf War veterans with GWI there was evidence of transmission of the illness. These families were not randomly chosen; they were families in which one or more veteran members were found to be positive for a mycoplasmal infection and one or more family members reported illnesses. We found that 57 out of 107 (53.2%) of these 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 CFS-symptomatic family members, most (40 out of 57 or 70.2%) had mycoplasmal infections compared to the few non-symptomatic family members who had similar mycoplasmal infections (6 out of 50 or 12%) (Table 5). When the incidence of mycoplasmal infection was compared within families, the CFS-symptomatic family members were more likely to have mycoplasmal infections compared to non-symptomatic family members (P<0.001). Although some non-symptomatic family members did have mycoplasmal infections (6 out of 50 or 12%), this was not significantly different from the incidence of mycoplasmal infections in healthy control subjects (6 out of 70 or 8.5%).
The mycoplasma infection types were also similar between GWI patients and their CFS-symptomatic family members. In 45 mycoplasma-positive CFS-symptomatic family members, most (31 out of 40 or 77.5%) had single species infections, similar to the mycoplasma-positive Gulf War veterans (37 out of 45 or 82%). Most of the species found in mycoplasma-positive GWI patients as well as in mycoplasma-positive family members with CFS were M. fermentans (Table 5). 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 (data not shown).
However, similar to previous reports, the time of onset of CFS illness after the Gulf War tended to be shorter in spouses than other family members, but these differences did not achieve significance (data not shown).
There is growing awareness that GWI is a not a unique, new syndrome caused by exposures that occurred during the Gulf War (5,9,21-24). GWI is characterized by a diverse collection of overlapping, persistent signs and symptoms from which several distinct subclasses of illness have been identified (3,4,21-24). Chronic infections are suspected as an important source of morbidity in a rather large subset of these patients (6-10,22-26). The most common infections found were intracellular mycoplasmal infections (6-10), but a few other chronic infections have been documented in GWI patients (25-27).
For example, parasitic infections, such as Malaria, Leishmaniasis and Schistosomiasis, have been found in some Gulf War veterans. These infections cause characteristic signs and symptoms and diagnostic tests are available (27). However, the prevalence of Leishmaniasis has been estimated at less than 100 cases in Gulf War veterans (28). Chronic infections may also cause other problems in GWI patients. Activation of the coagulation system seen in GWI patients could be related to chronic infections that cause vasculitis and coagulation disturbances (29).
In addition, approx. one-half of GWI patients were found to have fragile chromosomes that are more easily degraded by cellular nucleases, resulting in release of characteristic nucleotide fragments (30). This might be due to the action of intracellular mycoplasmas that are known to release chromosome-damaging chemicals (31).
Similar to previous results (6-10), mycoplasmal infections were found in ~41% of GWI patients. This has now been confirmed in a U. S. Department of Veterans’ Affairs study of 1,600 veterans (28). Although mycoplasmal infections were previously thought to produce relatively mild diseases limited to particular tissues or organs, such as urinary tract or respiratory system (13,14,28), the intracellular mycoplasmas detected in GWI patients with molecular techniques, primarily Mycoplasma fermentans, are highly virulent, colonize a wide variety of organs and tissues, and are difficult to treat (13,14). Such intracellular mycoplasmal infections have been successfully treated with long-term antibiotics, such as doxycycline, along with immune and nutritional support (6-9,16).
Although the results presented here document that the chronic infections found in Gulf War veterans with GWI can be found in symptomatic family members, we cannot extrapolate our results to the entire GWI patient population or their family members. First, our patient sample was not randomly selected. The presence of a positive mycoplasma test result on a veteran with GWI who reported illness in his/her immediate family formed the criteria for inclusion in the study.
Although CFS illnesses in immediate family members were commonly seen in our study, which examined families of mycoplasma-positive GWI patients, these illnesses are expected to be more difficult to find in the general GWI population where chemical, radiological and environmental exposures probably account for the majority of cases. Non-biological exposures should not be transmitted to family members, so studies on the entire GWI population may not yield significant results on transmission of illnesses to family members. Second, GWI patients and their family members were recruited from veterans groups, word of mouth, physician referrals and the Institute for Molecular Medicine website (www.immed.org); they were not recruited from specific military units.
Therefore, information on presumed exposures of veterans with respect to unit locations cannot be determined. Testing was performed using coded samples. After testing, all patients and control subjects completed patient Illness Survey Forms. Although some of these patients were examined by physicians at our associated clinics, most were seen by their own private physicians. Fourth, the validity of PCR techniques for Mycoplasma species detection has been questioned. In our studies, however, the sensitivity and specificity of the PCR method for Mycoplasma species detection were determined by examining serial dilutions of purified DNA from M. fermentans, M. pneumoniae, M. hominis and M. genitalium.
The primers produced the expected amplification product size in all test species, which was confirmed by hybridization using the appropriate 32P-labeled internal probe. Amounts as low as a few fg of purified DNA were detectable for all species with the specific internal probes. There was no cross-reactivity between the internal probes of one species and the PCR product from another species. In other experiments Mycoplasma species were added to whole blood at various concentrations. Specific PCR products down to 10 ccu/ml of blood could be detected (20).
Symptomatic family members of GWI patients were diagnosed with CSF/ME or a related fatiguing illness, Fibromyalgia Syndrome (FMS). In other studies, CFS and/or FMS patients were examined for systemic mycoplasmal infections, and about 50-60% of these patients were positive for any species of mycoplasma (9,19,20,32-34), indicating another link between mycoplasma-positive GWI patients and CFS-symptomatic family members. In contrast to mycoplasma-positive GWI patients and their mycoplasma-positive family members with CFS, several species of mycoplasmas in addition to M. fermentans were found in CSF/ME and FMS from non-military families (19,34). This further supports the hypothesis that mycoplasmal infections were transmitted from GWI patients to immediate family members who then presented with CFS/ME and for the most part had the same species of mycoplasmal infection.
The source of the mycoplasmal infections in GWI patients remains undetermined (28,35). A possible source for GWI patients is the multiple vaccines that were administered during the time of deployment to the Persian Gulf. A strong association has been found between GWI and the multiple vaccines that were administered (36-38). For example, Steele (4) found a three-fold increased incidence of GWI in non-deployed veterans who had been vaccinated in preparation for deployment, compared to non-deployed, non-vaccinated veterans, and Mahan et al. (38) found a two-times higher incidence of GWI signs and symptoms in veterans who recalled receiving anthrax vaccinations versus those who thought they had not.
Although the mycoplasmal infections found in GWI patients could have come from several sources, including offensive Biological Warfare attacks (35), we consider the most likely sources of the mycoplasmal infections in GWI patients were the multiple vaccines administered during deployment. Indeed, the signs and symptoms that have developed in Armed Forces personnel who recently received the anthrax vaccine are similar to those found in GWI patients. On some military bases this has resulted in chronic illnesses in as many as 7-10% of personnel receiving the vaccine (12,39).
Although the chronic signs and symptoms associated with anthrax vaccination are similar to those found in GWI patients, it is unlikely that all of the chronic illnesses reported by Gulf War veterans were caused by vaccines (39).
Undetectable microorganism contaminants in vaccines could have resulted in illness, and this may have been more likely in individuals with compromised immune systems caused by chemical and other exposures (28). Contamination with mycoplasmas has been found in commercial vaccines (40). Thus the vaccines used in the Gulf War should be considered as a possible source of the chronic infections found in mycoplasma-positive GWI patients and by airborne transmission in their mycoplasma-positive, CFS-symptomatic family members.
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