Download no serologic evidence of an association found between gulf war

Am. J. Trop. Med. Hyg., 60(5), 1999, pp. 752–757
Copyright q 1999 by The American Society of Tropical Medicine and Hygiene
NO SEROLOGIC EVIDENCE OF AN ASSOCIATION FOUND BETWEEN GULF WAR
SERVICE AND MYCOPLASMA FERMENTANS INFECTION
GREGORY C. GRAY, KEVIN S. KAISER, ANTHONY W. HAWKSWORTH, AND HAROLD L. WATSON
Emerging Illness Division, Department of Health Sciences and Epidemiology, Naval Health Research Center, San Diego,
California; Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama
Abstract. Occult occupational infection with Mycoplasma fermentans has been proposed as a cause for illness
among Persian Gulf War veterans. Symptom data and sera from a 1994–1995 cross-sectional survey of Navy Seabees
were used to select symptomatic and asymptomatic Gulf War veterans and nondeployed veterans to evaluate this
hypothesis. Survey sera from 96 Seabees were matched to prewar (before September 1990) archived sera. Immunoblot
serologic analyses were performed for M. fermentans in a controlled, blinded fashion. Both Gulf War veterans and
nondeployed veterans had prewar and postwar serologic evidence of M. fermentans infection consistent with natural
infection data. Among study subjects collectively, and stratified by Gulf War service, none of the immunoblot banding
profiles (prewar or postwar) or their changes over time were associated with postwar symptoms. These serologic data
do not support the hypothesis that Gulf War veterans have experienced Gulf War-related morbidity from M. fermentans
infection.
There has been much speculation and concern1–6 that the
increased reporting of symptoms7–9 by veterans of the Persian Gulf War may be due to a mysterious infectious disease
process. The Gulf War theater,10 located on and about the
Arabian Peninsula, was a desert environment with numerous
recognized infectious disease threats11,12 foreign to many of
the 697,000 deployed U.S. military personnel. Additionally,
Iraq had an extensive biological and chemical weapon development program and had used weapons of mass destruction domestically and in a war with Iran.13–16
Aggressive preventive measures,17,18 and the deployment
of a sophisticated field laboratory19 in the theater of operations provided substantial infectious disease morbidity data
during the conflict, which essentially ended in 1991.17,19–26
These data reveal that U.S. forces experienced diarrheal,21–24
respiratory,24,27 and parasitic infections,27 although the incidence was much lower than expected.17 Additional medical
data were collected after the war, when the Departments of
Defense and Veterans Affairs conducted more than 100,000
comprehensive clinical evaluations of veterans and their
families.5,28 However, other than the 12 U.S. Gulf War veterans who developed viscerotropic leishmaniasis,6,29–32 no
other new infectious disease processes have been recognized
as explanations for increased symptom reporting.4–6,33 Although there has been much discussion about Iraq’s weapon
program,16,34 expert review panels have agreed that it is unlikely that Iraq used biological weapons in the Gulf War.3,35
In addition to endemic infectious disease and biological
weapon threats, other infectious disease hypotheses have
been speculated, including Epstein-Barr virus infections,36
mycotoxin poisoning,37 cryptic streptococcal infection,4 and
microsporidia infection.4 Perhaps the most provocative of
these speculations has been the suggestion that a genetically
altered form of Mycoplasma fermentans was clandestinely
used as a biological agent during the Persian Gulf War.38–44
Allegations of U.S. government involvement in the purported biological agent’s development have astounded and concerned the public.45–47 These reports have won a number of
veteran advocates and have spawned a cottage industry of
special laboratory testing (classified as research testing and
not regulated) to aid Gulf War veterans in their diagnosis
and treatment.
The purpose of this research was to test the hypothesis
that symptomatic Gulf War veterans have serologic evidence
of M. fermentans infection.
SUBJECTS, MATERIALS AND METHODS
Study population and paired sera selection. The study
population and symptom analyses are described elsewhere.9
Briefly, during 1994 and 1995, a cross-sectional survey of
Navy construction workers (Seabees) who had remained on
active duty since the Persian Gulf War was performed. After
granting informed consent, 527 Gulf War veterans and 970
nondeployed veterans from 14 Seabee commands participated. In addition to answering questions regarding present and
past symptoms, participants donated sera that was matched
to banked (before September 1990) sera obtained from an
Army-Navy human immunodeficiency virus serum repository. Many of the study subjects, especially those who had
served in the Gulf War theater, complained of chronic nonspecific symptoms since the Gulf War.
Subjects were studied for the self-reported prevalence of
one month or more (since July 1990) of 46 symptoms. For
the purposes of this study, participants were screened by
their responses to seven of these symptom questions that
were most consistent with clinical reports of mycoplasmal
infection: cough, sore throat, pains that move from one joint
to another, joint swelling, joint redness, rash or skin ulcer,
fever, and asthma.48–50 After selecting the most symptomatic
subjects first and other subjects with a random-number procedure, 90 Gulf War veterans (60 symptomatic) and 60 nondeployed veterans (30 symptomatic) were identified. Only
those whose prewar sera were available were studied for
evidence of M. fermentans infection: symptomatic Gulf War
veterans (n 5 46), asymptomatic Gulf War veterans (n 5
18), symptomatic nondeployed veterans (n 5 20), and
asymptomatic nondeployed veterans (n 5 12). Three of these
96 Seabees were women. This study was conducted in accordance with the Department of the Navy’s Protection of
Human Subjects guidelines.
Laboratory procedures. Sera were shipped on dry ice to
the University of Alabama Diagnostic Mycoplasma Laboratory (Birmingham, AL). Laboratory staff were blinded
752
753
GULF WAR VETERANS AND M. FERMENTANS
with respect to each patient’s Gulf War experience and postwar symptoms.
Antigen preparation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and Western blot analysis were
performed as described elsewhere.51 Briefly, proteins from
M. fermentans strains incognitus, DEB, 17065, MT-2, K-7,
PG-18, and AOU were separated by electrophoresis using
10% resolving and 4% stacking gels. Separated proteins
were transferred for immunoblotting to nitrocellulose sheets
(Bio-Rad, Hercules, CA) according to the method of Towbin
and others.52 Immunologic reactions using patient serum diluted 1:100 were visualized with peroxidase-labeled conjugates (Sigma, St. Louis, MO). Sera were classified as immunoblot negative if no reactive antigens were detected and
as immunoblot positive if at least one antigen was detected.
Reactions were subjectively scored as weak positive based
on the intensity of the reaction relative to a positive reaction
(Figure 1). For statistical analyses, only positive reactions
were considered evidence for M. fermentans infection. If a
subject’s prewar sera was negative or weak and his postwar
survey sera was positive, he was considered to have evidence of a new M. fermentans infection since the war. Positive reactions were further stratified into reactions positive
with 30-kD antigens, 60-kD antigens, both 30-kD and 60kD antigens, or a mixture of three or more different antigens.
As a possible source of confounding for the M. fermentans
assays, the serologic responses (IgM and IgG) to M. pneumoniae were also determined using an ELISA.53–55 A whole
cell lysate antigen was used to maximize sensitivity. Positive
and negative controls were also used. A positive M. pneumoniae serologic result was defined as either a four-fold increase in IgM or IgG titers or a change in IgM titer from
negative to elevated. A subject’s IgM response greater than
the threshold value of 1:10 (derived from a group of control
subjects’ sera) was considered elevated.
Statistical analyses. Univariate risk factor associations
were examined using the chi-square test or Fisher’s exact
test. Exact confidence intervals56 were calculated about unadjusted odds ratios.
RESULTS
Gulf War veterans and nondeployed veterans were similar
with respect to race, marital status, gender, postwar hospitalizations, and symptom reporting (Table 1). However, Gulf
War veterans were slightly younger and less well educated
than their nondeployed peers. These findings were consistent
with findings from other studies of morbidity among Gulf
War veterans.57,58
Examples of the immunoblot analyses are shown in Figure
1. Variations of the antibody reactivity patterns included no
reaction for prewar or postwar sera, reaction for either prewar or postwar sera, and reactions for both prewar and postwar sera. The positive examples included in Figure 1 show
paired sera that demonstrated some degree of reaction from
both prewar and postwar sera.
Immunoblot testing of paired sera revealed that seven
(10.9 %) of 64 Gulf War veterans and three (9.3%) of 32
nondeployed had prewar antibody against M. fermentans.
Serologic testing of the study subjects without prewar antibody revealed that 11 (19.2%) of the 57 Gulf War veterans
and four (13.7%) of the 29 nondeployed veterans had serologic evidence of new M. fermentans infection since the war
(Table 2). Given these serologic results, individuals were
then examined by symptom reporting. Four (14.2%) of 28
asymptomatic subjects (both Gulf War veterans and nondeployed veterans) and 11 (19.0%) of 58 symptomatic subjects
had evidence of new M. fermentans infection (Table 2). We
then stratified by deployment status and symptom reporting.
Three (17.6%) of 17 asymptomatic Gulf War veterans and
one (9.1%) of 11 asymptomatic nondeployed veterans demonstrated serologic evidence of new M. fermentans infection
(Table 3). Similarly, eight (20%) of 40 symptomatic Gulf
War veterans and three (16.7%) of 18 symptomatic nondeployed veterans had serologic evidence of new M. fermentans infection (Table 3). None of these comparisons was statistically significant.
Additionally, among study subjects collectively, and stratified by Gulf War service, none of the specific immunoblot
banding profiles (prewar or postwar) or their changes over
time were statistically associated with postwar symptoms.
Finally, there was little indication that M. fermentans serologic analyses were confounded by M. pneumoniae infection. Only one individual (asymptomatic nondeployed veteran) had evidence of new infection with both M. fermentans
and M. pneumoniae over the time period of study.
DISCUSSION
Although M. fermentans has been recovered from the urogenital and respiratory tract, bone marrow, and joints of patients,59–62 little consideration was given to the possibility
that this organism was a human pathogen until the description of an organism tentatively named M. incognitus. It was
originally reported in patients with acquired immunodeficiency syndrome (AIDS) and was later found in non-AIDS
patients.49,63,64 Genetic and serologic comparisons subsequently confirmed that the organism originally designated as
M. incognitus was a strain of M. fermentans.65
Nicolson and Rosenberg-Nicolson have suggested that
mycoplasmas might be the cause of postwar morbidity
among Gulf War veterans.38 Since then, Nicolson and Nicolson have reported identifying occult mycoplasmal infection with a gene-tracking and forensic polymerase chain reaction technique and successfully treating symptomatic veterans with long courses of doxycycline and other antibiotics.66 They suggest that Gulf War veterans are transmitting
mycoplasmas via close contact with their family members.
They argued that the pathogen, M. fermentans (incognitus
strain), is intracellular and often fails to cause a humoral
immunogenic response, and that only their laboratory and
properly trained laboratories can detect the pathogen.67 Although validation studies are in progress, these experimental
laboratory procedures have not yet been properly corroborated and the efficacy of clinical treatments have not been
evaluated with appropriate clinical trials.
Mycoplasmal organisms cause a wide range of diseases,
are suspected in others, are particularly aggressive in immunocompromised individuals, and the infections are characteristically chronic. Due to the difficulty of detection by
culture, these organisms are likely to be grossly underdiagnosed. At least some individuals may harbor the organisms
754
GRAY AND OTHERS
FIGURE 1. Representative immunoblot analyses of prewar and postwar sera reactivity with Mycoplasma fermentans antigens. Proteins (20
mg) from each of the seven M. fermentans strains separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to
nitrocellulose. Immunologic reactions were visualized using peroxidate-labeled conjugates. The seven strains were loaded in order, left to right:
incognitius, DEB, 17065, MT-2, K-7, PG-18, and AOU. Identical panels were reacted with the prewar (left side of panel) and postwar (right
side of panel) sera from each patient. Five different paired sera are shown representing no reaction or reactions with antigens of 30-kD, 60kD, or multiple sizes. The approximate molecular mass of each reaction product(s) for each different serum is noted above each panel.
with little or no signs of illness. Furthermore, there often is
a remarkable degree of variability in pathogenicity among
different strains of the same organism.68 In most instances,
when disease does occur, the host response is an integral
part of lesion development. With the exception of certain
diseases of animals, mycoplasmal diseases are rarely fatal
and often clinically silent.68 Conversely, the host is seldom
capable of quickly eliminating the organism.68, 69 For example, M. pulmonis infection of rodents can persist for the life
of the host even in the presence of an intense inflammatory
and immune response and it ultimately may result in chronic
obstructive lung disease and contribute to death.68 In hu-
755
GULF WAR VETERANS AND M. FERMENTANS
TABLE 1
Demographic characteristics of study subjects by deployment status
Characteristic
Mean age (years)
Race (%)
White
Black
Other
Currently married (%)
Gender (%)
Male
Female
Education (%)
High school only
. High school
Hospitalized since July 1990 (%)
None
One or more
Symptoms for one month since July
1990 (%)
No symptoms
Two or more symptoms
Gulf War
veterans
(n 5 64)
Nondeployed
veterans
(n 5 32)
P
27.7
30.4
0.06*
81.3
7.8
10.9
65.6
74.2
12.9
12.9
87.1
0.68†
0.08†
96.9
3.1
96.9
3.1
1.00†
60.0
40.0
31.2
68.8
0.01†
58.3
41.7
71.9
28.1
0.20†
28.1
71.9
37.5
62.5
0.35†
* By t-test.
† By chi-square test.
mans, Ureaplasma urealyticum infection can persist in the
joint synovial fluid of immunocompromised individuals for
up to two years producing a slowly progressive, chronic arthritis.65
Animal model studies have shown that stress from environmental factors and microbial synergy can result in exacerbation of infection to a clinically significant disease. The
percentage of clinically insignificant human infections that
may respond similarly to stress factors is unknown. Thus, in
a population that is inherently stressed by a multitude of
physical and psychologic factors, such as military troops in
a war zone, it would not be unexpected to find that such
organisms play a significant role in the production of various
clinical symptoms.
Since mycoplasma infections are so often clinically silent
it is of importance to evaluate antibody responses in patients
with and without disease. This will help to discern if the
quality of an anti-mycoplasmal antibody response is reflective of active disease or, conversely, if one antibody response
is protective compared with another. This type of analysis is
most easily performed using immunoblot analyses since specific reactive antigens can be discerned, whereas ELISA
techniques do not currently have such precision. Here we
have examined reactions to numerous specific M. fermentans
antigens for associations with postwar symptoms. It is inter-
esting in this study that for serum pairs where there was
some reaction in both prewar and postwar sera, the antigens
recognized were the same. This may indicate that the immune systems of certain individuals are primed to respond
to specific antigens.
Although our analyses provide evidence that M. fermentans does not have a significant role in the illnesses reported
by Gulf War veterans, our findings must be interpreted with
caution. Our statistical power to detect small differences was
limited by the sample size. For example, using commonly
accepted parameters (a 5 0.05, b 5 0.20), we had only
enough subjects to detect an odds ratio of approximately 5.0
when we compared all Gulf War veterans (n 5 57) with
nondeployed veterans (n 5 29) for seroconversions. Such
power would have been quite adequate had we detected what
was suggested by others since there were almost no seroconversions among nondeployed veterans and many seroconversions among Gulf War veterans.67 Additionally, our
data are limited in that due to small sample sizes, we could
not adjust for potential confounders, such as age. However,
except for age and education, the study populations were
homogeneous with respect to demographic characteristics
and other potential confounders (Table 1). We argue that the
small difference in age is not likely to be biologically important.
Because of the long time period between paired sera and
the lack of clinical and antibiotic use data, our serologic
results likely underestimate the true incidence of infection.
However, if Gulf War veterans had postwar symptoms due
to active M. fermentans infection, one would expect to detect
a higher prevalence of positive reactions among postwar sera
for the most symptomatic Gulf War veterans. This has been
alleged by a few scientists, citing positive gene-tracking results in 14 (46.7%) of 30 symptomatic Gulf War veterans in
an uncontrolled study.67 Our data demonstrate no such high
postwar prevalence of M. fermentans reactions, nor statistically important changes in immunoblot reaction over time
among symptomatic or asymptomatic Gulf War veterans.
Much uncertainty remains concerning the etiology of
postwar symptoms among Gulf War veterans. Certainly,
there is a marked difference between Gulf War veteran and
nondeployed veteran symptom reporting.7–9 This difference
is likely due to multiple causes, including postwar stress.6,70
However, we agree with expert panels that doubt the M.
fermentans hypothesis as a cause for increased postwar morbidity.4,6,71
These data are consistent with several large epidemiologic
studies of postwar hospitalizations, which have not found
evidence of emerging infectious disease.57,58 Our findings are
TABLE 2
Odds ratios for serologic evidence of new Mycoplasma fermentans infection among Gulf War veterans and nondeployed veterans of the same era
Characteristic
Category
Number*
Number (%) with
new infection
Odds ratio
(95% confidence interval)†
Deployment
Gulf War veteran
Nondeployed veteran
57
29
11 (19.2)
4 (13.7)
1.49 (0.39–7.08)
Reference
Number of symptoms lasting
for at least one month
Two or more symptoms
No symptoms
58
28
11 (19.0)
4 (14.2)
1.40 (0.36–6.67)
Reference
* Numbers reflect subjects without prewar serologic evidence of M. fermentans infection.
† Exact confidence interval.
756
GRAY AND OTHERS
TABLE 3
Odds ratios for serologic evidence of new Mycoplasma fermentans infection among Gulf War veterans and nondeployed veterans of the same
era, stratified by symptom reporting
Category
Number
Symptomatic Gulf War veteran
Symptomatic nondeployed veteran
Asymptomatic Gulf War veteran
Asymptomatic nondeployed veteran
40
18
17
11
Number (%)
with new infection
8
3
3
1
(20.0)
(16.7)
(17.6)
(9.1)
Odds ratio
(95% confidence interval)*
1.25 (0.3–8.3)
Reference
2.1 (0.1–123.6)
Reference
* Exact confidence interval.
important to Gulf War veterans, providing evidence that M.
fermentans infections occurred both before and after the war,
and that these infections occur in military populations independent of deployment to the Persian Gulf theater.10 These
serologic data do not support the hypothesis that Gulf War
veterans have experienced Gulf War–related morbidity from
M. fermentans infection.
Acknowledgment: We thank Dr. Gary D. Gackstetter (Uniformed
Services University of the Health Sciences, Bethesda, MD) for most
helpful assistance and recommendations in conducting this research.
Financial support: This represents report no. 98–5, supported by the
Navy Medical Research and Development Command (Bethesda,
MD) under DoD/HA reimbursable - 6423.
Disclaimer: The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, the Department of Defense, or the U.S. Government.
Authors’ addresses: Gregory C. Gray, Kevin S. Kaiser, and Anthony
W. Hawksworth, Emerging Illness Division, Naval Health Research
Center, PO Box 85122, San Diego, CA 92186–5122. Harold L. Watson, Lilly Research Laboratories, Indianapolis, IN 46285.
Reprint requests: Gregory C. Gray, Emerging Illness Division, Naval
Health Research Center, PO Box 85122, San Diego, CA 92186–
5122.
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