Mycoplasmal Infection/FMS

[Guest guest]

Iris, this is very, very interesting. I'm still not sure what

conclusions to make, but I've stored this information in the back of my

mind for future reference.

[ ] Mycoplasmal Infection/FMS

> This article was referenced in this weeks Immune

> Support Weekly. I hope this is one that you haven't

> seen.

>

>

>

> High Prevalence of Mycoplasmal Infections in

> Symptomatic (Chronic Fatigue Syndrome) Family Members

> of Mycoplasma-Positive Gulf War Illness Patients

>

>

> ImmuneSupport.com

> 01-01-2003

> Garth L. Nicolson,(1) PhD, Marwan Y. Nasralla,2 PhD,

> 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: gnicolson@...

>

> SUMMARY

> 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

>

> INTRODUCTION

> 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 ative 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

>

> Patients

>

> 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 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). Statistics

>

> 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.

>

> RESULTS

> As found in previous studies (9,16), veterans of the

> Gulf War with chronic illnesses (GWI) exhibited

> multiple signs and symptoms (Figures 1A, . 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,

> . 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, .

>

> 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).

>

> DISCUSSION

> 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.