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


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. REFERENCES

1. National Institute of Health, 1994. National Institutes of Health Technology Assessment Workshop Panel: the Persian Gulf experience and health. JAMA 272: 391–396. 2. Persian Gulf Veterans Coordinating Board, 1995. Unexplained illnesses among Desert Storm veterans: a search for causes, treatment, and cooperation. Report of the Persian Gulf Veterans Coordinating Board. Arch Intern Med 155: 262–268. 3. Institute of Medicine, 1995. Health Consequences of Service During the Persian Gulf War: Initial Findings and Recommendations for Immediate Action. Washington, DC: National Academy Press. 4. Institute of Medicine, 1996. Health Consequences of Service During the Persian Gulf War: Recommendations for Research and Information Systems. Washington, DC: National Academy Press. 5. Presidential Advisory Committee on Gulf War Veterans’ Illnesses, 1996. Interim Report, February 1996. Washington, DC: U.S. Government Printing Office. 6. Presidential Advisory Committee on Gulf War Veterans’ Illnesses, 1996. Final Report, December 1996. Washington, DC: U.S. Government Printing Office. 7. The Iowa Persian Gulf Study Group, 1997. Self-reported illness and health status among Persian Gulf War veterans: a population-based study. JAMA 277: 238–245. 8. U.S. Department of Health and Human Services, 1995. Unexplained illness among Persian Gulf veterans in an Air National Guard Unit: preliminary report - August 1990–March 1995. MMWR Morb Mortal Wkly Rep 44: 443–447.

9. Gray GC, Kaiser KS, Hawksworth AW, Hall FW, Barrett-Connor E, 1999. Increased postwar symptoms and psychological morbidity among U.S. Navy Gulf War veterans. Am J Trop Med Hyg 60: 758–766. 10. 1991. Executive Order 12744: Designation of Arabian Peninsula areas, airspace and adjacent waters as a combat zone. Fed Register 56 (15). 11. Oldfield EI, Wallace M, Hyams K, Yousif A, Lewis D, Bourgeois A, 1991. Endemic infectious diseases of the Middle East. Rev Infect Dis 13: 199–217. 12. Gasser RJ, Magill A, Oster C, Tramont E, 1991. The threat of infectious disease in Americans returning from Operation Desert Storm. N Engl J Med 324: 859–864. 13. Gertz B, 1996. Horror weapons. Air Force Magazine 79: 44– 48. 14. Adler T, 1994. Desert Storm’s medical quandary: do Iraqi chemical and biological agents explain Gulf War syndrome? Sci News 145: 394–395. 15. Dickey C, 1995. His secret weapon. Iraq: Saddam had a big germ-warfare arsenal. Newsweek (August 4): 34. 16. Zilinskas R, 1997. Iraq’s biological weapons. JAMA 278: 418– 424. 17. Hyams K, Hanson K, Wignall F, Escamilla J, Oldfield E, 1995. The impact of infectious diseases on the health of U.S. troops deployed to the Persian Gulf during Operations Desert Shield and Desert Storm. Clin Infect Dis 20: 1497–1504. 18. Richards A, Hyams K, Merrell B, Dasch G, Woody J, Ksiazek T, LeDuc J, Watts D, Cohen K, Goodnough C, Levine D, Blanck R, Bell W, 1991. Medical aspects of Operation Desert Storm. N Engl J Med 325: 970. 19. Hyams K, Bourgeois A, Escamilla J, Burans J, Woody J, 1993. The Navy forward laboratory during Operations Desert Shield/Desert Storm. Mil Med 158: 729–732. 20. Cope S, Schultz G, Richards A, Savage H, Smith G, Mitchell C, Fryauff D, Conlon J, Corneil J, Hyams K, 1996. Assessment of arthropod vectors of infectious diseases in areas of U.S. troop deployment in the Persian Gulf. Am J Trop Med Hyg 54: 49–53. 21. Hyams K, Bourgeois A, Merrell B, Rozmajzl P, Escamilla J, Thornton S, Wasserman G, Burke A, Echeverria P, Green K, Kapikian A, Woody J, 1991. Diarrheal disease during Operation Desert Shield. N Engl J Med 325: 1423–1428. 22. Hyams K, Malone J, Kapikian A, Estes M, Xi J, Bourgeois A, Paparello S, Hawkins R, Green K, 1993. Norwalk virus infection among Desert Storm troops. J Infect Dis 167: 986– 987. 23. Hyams KC, Malone JD, Bourgeois AL, Hawkins R, Hale TL, Murphy JR, 1995. Serum antibody to lipopolysaccharide antigens of Shigella species among U.S. military personnel deployed to Saudi Arabia and Kuwait during Operations Desert Shield and Desert Storm. Clin Diagn Lab Immunol 2: 700– 703. 24. Paparello S, Bourgeois A, Garst P, Hyams K, 1993. Diarrheal and respiratory disease aboard the hospital ship, USNS Mercy T-AH 19, during Operation Desert Shield. Mil Med 158: 392– 395. 25. Richards A, Malone J, Sheris S, Weddle J, Rossi C, Ksiazek T, Leduc J, Dasch G, Hyams K, 1993. Arbovirus and rickettsial infections among combat troops during Operation Desert Shield/Desert Storm. J Infect Dis 168: 1080–1081. 26. Richards A, Hyams K, Watts D, Rozmajzl P, Woody J, Merrell


27. 28. 29. 30. 31.

32. 33.

34.

35.

36. 37. 38. 39.

40. 41. 42. 43. 44. 45. 46. 47. 48.

49.

50.

B, 1993. Respiratory disease among military personnel in Saudi Arabia during Operation Desert Shield. Am J Public Health 83: 1326–1329. Malone J, Paparello S, Thornton S, Mapes T, Haberberger R, Hyams K, 1991. Parasitic infections in troops returning from Operation Desert Storm. N Engl J Med 325: 1448–1449. Joseph S, The Comprehensive Clinical Evaluation Program Evaluation Team, 1997. A comprehensive clinical evaluation of 20,000 Persian Gulf War veterans. Mil Med 162: 149–155. Magill A, Gasser RJ, Oster C, Grogl M, Sun W, 1992. Viscerotropic leishmaniasis in persons returning from Operation Desert Storm 1990–1991. Arch Dermatol 128: 1033–1034. Magill A, Grogl M, Gasser R, Oster C, 1993. Visceral leishmaniases in Desert Storm veterans. N Engl J Med 329: 1504. Magill A, Grogl M, Johnson S, Gasser R, 1994. Visceral infection due to Leishmania tropica in a veteran of Operation Desert Storm who presented 2 years after leaving Saudi Arabia. Clin Infect Dis 19: 805–806. Magill A, Grogl M, Gasser R, Sun W, Oster C, 1993. Visceral infection caused by Leishmania tropica in veterans of Operation Desert Storm. N Engl J Med 328: 1383–1387. Fukuda K, Nisenbaum R, Stewart G, Thompson WW, Robin L, Washko RM, Noah DL, Barrett DH, Randall B, Herwaldt BL, Mawle AC, Reeves WC, 1998. Chronic multisymptom illness affecting Air Force veterans of the Gulf War. JAMA 280: 981– 988. Tucker J, 1996. Saddam’s toxic plot: did Iraqi military planners hatch a secret scheme to debilitate our troops with low levels of chemical and biological weapons? Am Legion 26–27: 50– 51. Defense Science Board, 1994. Final Report: Defense Science Board Task Force on Persian Gulf War Health Effects. Washington, DC: Office of the Under Secretary of Defense for Acquisition and Technology. Carver L, Flanigan S, Connallon P, Crossley-Miller M, 1994. Epstein-Barr virus infection in Desert Storm reservists. Mil Med 159: 580–582. Tucker JB, 1997. Mycotoxins and Gulf War Illness: A Possible Link. Washington, DC: Insignia Publishing Company. Nicolson G, Rosenberg-Nicolson N, 1995. Doxycycline treatment and Desert Storm (letter). JAMA 273: 618–619. Nicolson GL, Hyman E, Korenyi-Both A, Lopez DA, Nicolson N, Rea W, Urnovitz H, 1995. Progress on Persian Gulf War illnesses–reality and hypotheses. Int J Occup Med Toxicol 4: 365–370. Nicolson GL, 1996. Gulf War illness: a bacterial infection. The Washington Post (January 23): A14. Nicolson G, 1996. Gulf War illness linked to fatigue syndrome. The New York Times (April 10): A18. Nicolson G, Bruton DJ, Nicolson N, 1996. Chronic fatigue illness and Operation Desert Storm. J Occup Environ Med 38: 14–16. Nicolson G, 1997. Cooperating on Gulf War illness. The Washington Post (January 25): A17. Nicolson G, Nicolson N, 1997. The eight myths of Operation ‘‘Desert Storm’’ and Gulf War syndrome. Med Confl Surviv 13: 140–146. Nicolson N, 1995. Massive cover-up of Desert Storm illness source. Criminal Politics (March): 32–36. Patterson R, 1996. The Case for Gulf War Gate. Houston Press (August 3–9). Fumento M, 1997. Gulf Lore Syndrome. Reason 28: 23–33. Krause DC, Taylor-Robinson D, 1992. Mycoplasmas which infect humans. Maniloff J, McElhaney RN, Finch LR, Baseman JB, eds. Mycoplasmas: Molecular Biology and Pathogenesis. Washington, DC: American Society for Microbiology, 417– 444. Lo SC, Dawson MS, Newton III PB, Sonoda MA, Shih JW, Engler WF, Wang RY, Wear DJ, 1989. Association of the virus-like infectious agent originally reported in patients with AIDS with acute fatal disease in previously healthy non-AIDS patients. Am J Trop Med Hyg 41: 364–376. Biberfeld G, 1985. Infection sequelae and autoimmune reactions

51. 52.

53.

54.

55.

56. 57.

58. 59. 60. 61. 62. 63.

64. 65. 66. 67. 68.

69. 70. 71.

757

in Mycoplasma pneumoniae infection. Razin R, Barile MR, eds. The Mycoplasmas. Orlando, FL: Academic Press, 293– 311. Stadtlander C, Zuhua C, Watson H, Cassell G, 1991. Protein and antigen heterogeneity among strains of Mycoplasma fermentans. Infect Immun 59: 3319–3322. Towbin H, Staehelin T, Gordon J, 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354. Cassell G, Drnec J, Waites K, Pate M, Duffy L, Watson H, McIntosh J, 1991. Efficacy of clarithromycin against Mycoplasma pneumoniae. J Antimicrob Chemother 27 (suppl A): 47–59. Cassell G, Gambil G, Duffy L, 1996. ELISA in respiratory infections of humans. Tully J, Razin S, eds. Molecular and Diagnostic Procedures in Mycoplasmology. Volume II. San Diego, CA: Academic Press, 123–136. Gray GC, Duffy LB, Paver RJ, Putnam SD, Reynolds RJ, Cassell GH, 1997. Mycoplasma pneumoniae: a frequent cause of pneumonia among U.S. Marines in southern California. Mil Med 162: 524–526. Mehta C, Patel B, Gray R, 1985. Computing an exact confidence interval for the common odds ratio in several 2 3 2 contingency tables. J Am Stat Assoc 80: 969–973. Gray GC, Coate BC, Anderson CM, Kang HK, Berg SW, Wignall FS, Knoke JD, Barrett-Connor E, 1996. The postwar hospitalization experience of U.S. veterans of the Persian Gulf War. N Engl J Med 335: 1505–1513. Knoke JD, Gray GC, 1998. Hospitalizations for unexplained illnesses among U.S. Persian Gulf War veterans. Emerg Infect Dis 4: 211–219. WIlliams MH, Brostoff J, Roitt IM, 1970. Possible role of Mycoplasma fermentans in pathogenesis of rheumatoid arthritis. Lancet 2: 277–280. Murphy WH, Bullis C, Ertel IJ, Zarafonetis JD, 1967. Mycoplasma studies of human leukemia. Ann N Y Acad Sci 143: 544–556. Barile M, 1967. Mycoplasma and leukemia. Ann NY Acad Sci 143: 557–572. Sasaki T, 1991. Evidence that mycoplasmas, gram-negative bacteria, and certain gram-positive bacteria share a similar protein antigen. J Bacteriol 173: 2398–2400. Bauer FA, Wear DJ, Angritt P, Lo SC, 1991. Mycoplasma fermentans (incognitus strain) infection in the kidney of patients with acquired immunodeficiency syndrome and associated nephropathy. Hum Pathol 22: 63–69. Lo SC, 1986. Isolation and identification of a novel virus from patients with AIDS. Am J Trop Med Hyg 35: 675–676. Vogler LB, Waites KB, Wright FP, Perrin JM, Cassell GH, 1985. Ureaplasma urealyticum polyarthritis in agammaglobulinemia. Pediatr Infect Dis 4: 687–691. Nicolson G, Nicolson N, 1996. Mycoplasmal infections–diagnosis and treatment of Gulf War illness/CFIDS. CFIDS Chronicle 9: 66–69. Nicolson G, Nicolson N, 1996. Diagnosis and treatment of mycoplasma infections in Persian Gulf War illness—CFIDS patients. Int J Occup Med Immunol Toxicol 5: 69–78. Simecka J, Davis J, Davidson M, Ross S, Stadtander C, Cassell G, 1992. Mycoplasma disease in animals. Maniloff J, McElhaney R, Finch L, Baseman J, eds. Mycoplasmas: Molecular Biology and Pathogenesis. Washington, DC: American Society of Microbiology, 391–415. Cassell G, Clyde Jr W, Davis J, 1985. Mycoplasma pathogenicity. Razin S, Barile M, eds. The Mycoplasmas. New York: Academic Press, 65–106. Hyams K, Wignall F, Roswell R, 1996. War syndromes and their evaluation: from the U.S. Civil War to the Persian Gulf War. Ann Intern Med 125: 398–405. Dybvig K, 1998. Mycoplasma and illness. United States Senate Committee on Veterans’ Affairs: Report of the Special Investigation Unit on Gulf War Illnesses. Washington, DC: US Government Printing Office, 216–225.