By Prof. Garth L. Nicolson The Institute for Molecular Medicine

This article was published in Fibromyalgia Frontiers (Vol. 10, No. 3, pp. 5-9 & 27-28, 2002), the official publication of The National Fibromyalgia Partnership (www.fmpartnership.org).

Fibromyalgia Syndrome (FMS), Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS) and Gulf War Illnesses (GWI) are characterized by overlapping chronic signs and symptoms. The characteristic difference is that FMS patients present with widespread pain, but this is sometimes seen in CFS/ME and GWI patients. This is why we originally proposed that GWI is a CFS/ME or in some patients a FMS- or CFS-like illness [1]. In addition, FMS patients often have a secondary diagnosis of CFS. Other chronic illnesses, such as Rheumatoid Arthritis (RA), Inflammatory Bowel Disease, among others, also show some of the same signs and symptoms in addition to their obvious differences, suggesting that there may be some overlap in the underlying causes of these chronic conditions or at least in the factors that may result in sickness (morbidity) or illness progression [2].

MICROORGANISMS CAUSE MORBIDITY IN MANY FMS, CFS AND GWI PATIENTS. Although the causes of chronic illnesses are for the most part unknown, the complex signs and symptoms that evolve in many, perhaps even a majority of patients, may be due, in part, to systemic chronic infections (bacteria, viruses, fungi). Such infections can follow acute or chronic chemical or other insults (viral, environmental, trauma, etc.) that have the potential to temporarily suppress the immune system and modify the endocrine system [2]. Chronic illnesses probably evolve over time as a multi-step process that may require additional, multiple toxic exposures, including infections that can be causative for the illness in some patients, cofactors for the illness (not causative but important to morbidity) in others or opportunistic in immune-compromised patients. Chronic infections that are usually held in check by our immune systems can take hold if they can avoid immune surveillance and penetrate and hide in various tissues and organs, including cells of the Central and Peripheral Nervous Systems. When such infections occur, they can cause many of the complex signs and symptoms seen in FMS, CFS, RA and GWI, including widespread pain and immune dysfunction [2].

Changes in environmental responses as (allergies, chemical sensitivities, etc.) as well as increased titers to various endogenous viruses that are commonly found to be expressed in these patients have been seen in FMS, CFS/ME, RA and GWI patients [2, 3]. Chronic infections have been found to be important cofactors in some illnesses, including HIV-AIDS and other immunodeficiency disorders, skin diseases and some autoimmune diseases [3]. It is proposed that autoimmune signs and symptoms are caused when intracellular pathogens, such as mycoplasmas and other bacteria, escape from cellular compartments. Such microorganisms can incorporate into their own structures pieces of host cell membranes that contain important host membrane antigens that can trigger autoimmune responses, and they can also produce products that mimic host cell antigen structures. Thus, patients with such infections may respond immunologically to microorganism antigens as well as their own membrane antigens, producing unusual autoimmune signs and symptoms. When such microorganisms colonize peripheral nerves, they can induce autoimmune attacks on nerve cells that cause chronic inflammation and even some destruction of nerve cells.

>B>BACTERIAL INFECTIONS IN FMS AND OTHER CHRONIC ILLNESSES Bacterial infections have been commonly found in FMS and other chronic illness patients [2, 3], and these infections may be important factors in the illness that require treatment. Although chronic illness patients like other patients are exposed to various bacterial pathogens, only certain bacterial species have the capacity to cause chronic signs and symptoms. As an example, one type of airborne microorganism that has received renewed interest of late as an important cause, cofactor or opportunistic infection in these disorders is represented by various primitive classes of bacteria [2]. These microorganisms, principally Mycoplasmas and other primitive bacteria (Chlamydia, Coxiella, Brucella, Borrelia, etc.), although not as well known as other agents in causing disease, are now considered important emerging pathogens in various chronic diseases where a majority of patients show evidence of these infections in their blood.

In our recent study on CFS and FMS patients, most patients had multiple bacterial infections, especially if they had been sick for many years or had severe signs and symptoms [4]. These infections invade the vascular system and cause coagulation problems, and they can cause or increase the risk of coronary diseases, such as endocarditis and myocarditis. We found that some patients have Borrelia, Brucella or Chlamydia infections. For example, most patients diagnosed with Borrelia burgdorferi infections (Lyme Disease) show the same signs and symptoms as CFS patients and some have FMS-like signs and symptoms. Although few urban patients have Brucella infections, these are more commonly found in rural patients. Chlamydia pneumoniae infections were found in approximately 7% of CFS or FMS patients and one control subject out of 100 that also did not have other bacterial or viral infections [5]. This finding is similar but somewhat lower than previously reported for CFS patients [6].

VIRAL INFECTIONS IN FMS AND OTHER CHRONIC ILLNESSES Another type of infection that appears to play an important role in various chronic illnesses is viral in origin. Different types of viruses have been found in CFS, FMS and various autoimmune diseases. One of the most common viral infections found in CFS patients is Human Herpes Virus-6 or HHV-6 [7, 8]. Antibodies against HHV-6 are routinely found in CFS patients, and most of these patients have the HHV-6A variant [9]. HHV-6 appears to play a role as a major contributing factor in several chronic illnesses [7, 8]. Although several studies have associated HHV-6 with CFS [9-13], there are also reports that could not find a HHV-6 association with CFS/ME [14, 15]. When we studied CFS or FMS patients we found that approximately 31% had HHV-6 infections [5].

Others have found even higher percentages of patients with HHV-6 infections [9-12]. In control subjects without evidence of signs or symptoms we found HHV-6 infections in 9 of 100 subjects. None of these HHV-6-positive control subjects had other infections [5]. In addition to HHV-6, cytomegalovirus or CMV has been found in chronic illness patients. CMV-like ""stealth virus"" has been identified in CFS/ME patients by isolation and nucleotide sequencing [16]. Similar viruses have also been found in bipolar patients and patients with acute encephalopathy [17]. Although the exact role that this type of virus plays is not known, this class of virus is known to cause neurological dysfunction, and they can produce molecules similar to known chemokine or chemical messenger molecules important in inflammation [18].

MULTIPLE CO- NFECTIONS IN CHRONIC ILLNESSES Previously we found multiple co-infections of various mycoplasma species in a majority of North American [4] and European [19] CFS and FMS patients. This occurred when one of the mycoplasma species was either M. fermentans or M. hominis. When we examined the incidence of active HHV-6 infections in mycoplasma-positive and negative patients, we found that there was no preference for HHV-6 infections in mycoplasma-infected patients. For example, we found that the incidence of HHV-6 in mycoplasma-positive patients was 30.7%, whereas in mycoplasma-negative patients HHV-6 infections were found in 30.2% of patients. There was also no preference for particular Mycoplasma species in HHV-6 co- infections [5]. We also examined the incidence of C. pneumoniae infections in mycoplasma-positive and negative patients and found that there was no preference for multiple infections, nor was there a preference for particular Mycoplasma species in C. pneumoniae+mycoplasma co-infections. In mycoplasma-positive patients C. pneumoniae infections were found in 7.7% of patients, whereas in mycoplasma-negative patients C. pneumoniae infections were found in 7.3% of patients. Similarly, in HHV-6-positive patients C. pneumoniae infections were found in 8.2% of patients, whereas in HHV-6- negative patients C. pneumoniae infections were found in 7.2% of patients [5]. Although we expected to find patients infected with different types of infectious microorganisms, an unexpected finding in our study was that Mycoplasma species, Chlamydia pneumoniae and HHV-6 infections were not clustered together in most patients. When we examined the incidence of Chlamydia pneumoniae or HHV-6 in mycoplasma-positive or negative patients, we that clustering of these infections is not more likely in certain patients. Although patients that had multiple infections had on the average more severe signs and symptoms, we did not find differences between the types of infections and signs and symptoms. Many of the signs and symptoms of FMS and CFS are relatively non-specific; therefore, the possibility of unique patient subsets with different patterns of signs/symptoms based on the types of infections present is probably unlikely and consistent with our laboratory results.

FAMILY STUDY SHOWS THAT SOME GWI PATIENTS HAVE A CONTAGIOUS ILLNESS Veterans of the Gulf War with chronic illnesses (Gulf War Illnesses or GWI) display multiple signs and symptoms [1]. Upon examination, the signs and symptoms of GWI were indistinguishable from civilian patients in the same immediate family diagnosed with CFS or FMS [1], expect for symptomatic children aged 3-12 who were also diagnosed with autism [20]. Similar to previous studies on GWI patients [21, 22], we found that 45 of 110 patients or ~42% had mycoplasmal infections , and almost all of these (37 out of 45 or ~82%) were single infections [20]. M. fermentans was found in ~85% of these single infection cases. When the few multiple species infection cases were examined, most were found to have combinations of M. fermentans plus either M. pneumoniae, M. hominis or M. genitalium. 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 [20]. In families of Gulf War veterans with GWI there was evidence of transmission of the illness to immediate family members. In our study the 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 or FMS (and Autism or Attention Deficit Hyperactivity Disorder in their children) [20]. Among 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%). When the incidence of mycoplasmal infection(s) was compared within families, the symptomatic family members were significantly more likely to have mycoplasmal infections compared to non-symptomatic family members. Symptomatic children (mostly diagnosed with Autism and other chronic disorders) in these families were also infected with mycoplasmas at high incidence, but this was not seen in aged-matched control subjects. 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 control subjects (6 out of 70 or 8.5%) [20]. Some of these control subjects may be carriers and some may eventually present with CFS/ME or other illnesses related to mycoplasmal infections. GWI patients had similar infections to their symptomatic family members. In 45 mycoplasma-positive symptomatic family members, most (77.5%) had single species infections, similar to the mycoplasma-positive Gulf War veterans (82%). Most mycoplasma-positive GWI patients as well as mycoplasma-positive family members or children diagnosed with Autism had M. fermentans infections. 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 [20].

POSSIBLE SOURCE OF MICROORGANISMS FOUND IN CHRONIC ILLNESS PATIENTS Most microorganisms are not considered as important human pathogens when they are found at superficial sites, such as the oral cavity or gut, but some species, such as certain species of mycoplasma, among others, have the capacity to penetrate into the blood circulation and colonize various tissues, and these cell-penetrating microorganisms have been associated with various human diseases [3]. Similarly, infections such as HHV-6 also penetrate the blood vascular where they colonize blood vessel endothelial cells. Do such infectious agents actually cause FMS or other chronic illnesses? Probably not on their own, but microorganisms like Mycoplasma, Chlamydia, Brucella, Coxiella and other bacteria and some viruses, in particular HHV-6 and CMV, appear to be important in causing chronic illness progression and patient morbidity, exacerbating the major signs and symptoms seen in patients with chronic illnesses. Our recent results have shown that the combination of multiple bacterial and viral infections can be particularly difficult for patients to overcome. How do patients contract the bacterial and viral infections found in FMS? The answer to this is not known, but many patients may already have these infections in dormant or latent forms, and their triggering of illness may be associated with a decline in the ability of the immune system to hold them in check. For example, many chronic illness patients report prior severe trauma or acute infections preceded their illness. In these patients, infections may occur by a variety of routes, including airborne transmission, fluid exchange (such as blood transfusions), or in contaminated vaccines. Only rarely are vaccines contaminated, and most vaccinations would not be expected to cause illness, but in some commercial vaccines contamination has been documented. For example, some of the more common contaminants of commercial vaccines are mycoplasmas, found in approximately 6% of commercial vaccines [23]. In the case of GWI, the multiple vaccines that the Armed Forces received upon deployment to the Persian Gulf have recently been associated with GWI. Unwin and colleagues [24] studied British Gulf War veterans with GWI and found an association with the multiple vaccines that they received during deployment. In the U.S. there has been a rash of GWI signs and symptoms in Armed Forces personnel who recently received the anthrax vaccine. In some cases this has resulted in chronic illnesses in as many as 7-10% of personnel receiving the vaccine [25]. The chronic signs and symptoms associated with anthrax vaccination are very similar to those found in GWI patients, suggesting that at least some of the chronic illnesses suffered by veterans of the 1991 Gulf War may have been caused by vaccines in combination with other exposures [25]. The anthrax vaccine, in particular, should not be used in Armed Forces personnel because of valid questions concerning its safety and efficacy.

ANTIBIOTIC AND ANTIVIRAL TREATMENTS FOR CHRONIC ILLNESSES When microorganism infections are identified in blood fractions of subsets of patients with FMS or other chronic illnesses, these patients can be treated as medical not psychological or psychiatric patients, just like any other patients with systemic bacterial infections. This does not mean that psychological or psychiatric problems are not important in some chronic illness patients. But if such infections are important in these disorders, then appropriate treatments with antibiotics, antivirals or other medications that suppress chronic infections should result in improvement and even recovery. Long-term treatment is required for the types of bacterial infections found in FMS and other chronic illnesses [26]. Few patients recover after only a few cycles of therapy, possibly because of the intracellular locations of the infections and the slow-growing nature of most of these microorganisms. We now recommend that patients who have been diagnosed with blood infections receive continuous antibiotics for at least 6 months before using 6-week cycles of treatment [24] Although patients starting such therapy usually have Herxheimer reactions and feel initially worse due to die-off or release of toxic materials from damaged microorganisms, they eventually stabilize and then slowly begin to recover. Unfortunately, the treatment requires long-term therapy and recovery is usually very slow. Patients that have been sick for many years are unlikely to recover within a year of therapy. We are also examining oxygen therapy using hyperbaric oxygen treatment. The clinical responses seen with antibiotics that are seen are not due to placebo effects, because administration of some antibiotics, such as penicillins to patients with mycoplasmal infections, resulted in patients becoming more not less symptomatic. In addition, they are not due to immunosuppressive effects of some of the antibiotics, because other antibiotics that do not cause immune suppression are also effective but only if they suppress the chronic infections. Some patients recover to a certain point and then fail to continue to respond to the recommended antibiotics, suggesting that other problems, such as viral infections, environmental exposures and other toxic events also play an important role in these illnesses, and may even play a predominant role in some patients [27]. Since many patients also have viral (HHV-6, CMV, etc.) infections, these must also be treated. For severe infections, there are some antivirals that can help, but most patients do well on immune enhancement and nutritional supplements (see www.immed.org for further information).

COMPLEX ROLE OF TOXIC EXPOSURES IN CHRONIC ILLNESSES Do chronic infections explain illnesses like FMS or CFS? It is unlikely that there are only one or even a few explanations for complex chronic illnesses. Rather, these illnesses are probably due to a combination of multiple toxic exposures, chemical and biological, in combination with genetic susceptibility (immune systems and/or detoxification systems) that determines whether a person becomes chronically ill. These considerations probably also play an important role in determining who will recover to various extents on different types of therapy. In addition, recovery can be complicated by patients'' over-dependence on drugs, such as certain antidepressants or other drugs that can suppress portions of the immune system. The treatments of chronic illnesses that are due to toxic exposures from chemical or radiological agents are quite different from the treatment of chronic infections [2]. The treatment of chemically exposed patients usually involves removal of offending chemicals from the patient's environment, depletion of chemicals from the patient's system and treatment of the signs and symptoms caused by chemical exposure(s). Chemically exposed patients are often extremely sensitive to a variety of commonly encountered chemicals, including perfumes and air fresheners, petrochemical fumes, chlorine, cleaning solutions and solvents, among others. They are also very sensitive to certain foods, and special diets are often necessary, and in some cases direct skin contact with certain substances can cause strong cutaneous reactions. Therefore, an important part of treatment for chemical exposures requires limiting exposures to a variety of common chemicals and gradual removal of the toxic chemical [28].

FOR FURTHER INFORMATION The Institute for Molecular Medicine and its certified reference diagnostic lab, International Molecular Diagnostics (www.imd-lab.com) can test patients for evidence of bacterial and viral infections of the types that are associated with chronic diseases like FMS, CFS, GWI and RA and autoimmune diseases. The website for further information is: www.immed.org. Contact: Prof. Garth L. Nicolson The Institute for Molecular Medicine (website: www.immed.org) 15162 Triton Lane, Huntington Beach, CA 92649-1401 Tel: 714-903-2900 Fax: 714-379-2082 email: gnicolson@immed.org

References
1. Nicolson GL, Nicolson NL. Chronic fatigue illness and Operation Desert Storm. J. Occup. Environ. Med. 1996; 38:14-16. [find article at www.immed.org]
2. Nicolson GL, Nasralla M, Hier J, et al. Mycoplasmal infections in chronic illnesses: Fibromyalgia and Chronic Fatigue Syndromes, Gulf War Illness, HIV-AIDS and Rheumatoid Arthritis. Med. Sentinel 1999; 4:172-176. [find article at www.immed.org]
3. Nicolson GL, Nasralla M, Franco AR, De Meirleir K, et al. Mycoplasmal infections in chronic diseases. J. Chronic Fatigue Syndr. 2000; 6(3/4):23-39. [find article at www.immed.org]
4. Nasralla M, Haier J, Nicolson GL. Multiple mycoplasmal infections detected in blood of Chronic Fatigue and Fibromyalgia Syndrome patients. Eur. J. Clin. Microbiol. Infect. Dis. 1999; 18:859-865. [find article at www.immed.org]
5. Nicolson GL, Nasralla M, De Meirleir K, Gan, Haier J. Evidence for bacterial (mycoplasma, Chlamydia) and viral (HHV-6) co-infections in chronic fatigue syndrome patients. J. Chronic Fatigue Syndr. 2002; 10: In press. [find article at www.immed.org]
6. Chia JKS, Chia LY. Chronic Chlamydia pneumoniae infection: a treatable cause of Chronic Fatigue Syndrome. Clin. Infect. Dis. 1999; 29:452-453.
7. Braun DK, Dominguez G, Pellett PE. Human herpesvirus-6. Clin. Microbiol. Rev. 1997; 10:521-567.
8. Campadelli-Fiume G, Mirandela P, Menetti L. Human herpesvirus-6: an emerging pathogen. Emerg. Infect. Dis. 1999; 5:353-366.
9. Patnaik M, Komaroff AL, Conley C, Orjin-Amaine EA, Peter JB. Prevalence of IgM antibodies to human herpesvirus-6 early antigen in patients with chronic fatigue syndrome. J. Infect. Dis. 1995; 172:1164-1167.
10. Wagner M, Krueger GRF, Ablashi DV, Whitman JE. Chronic fatigue syndrome (CFS): a critical evaluation of testing for active human herpesvirus-6 (HHV-6) infection: a review of data on 107 cases. J. Chronic Fatigue Syndr. 1996; 5:3-16.
11. Zorsenon M, Colle R, Rukh G et al. Active HHV-6 infection in Chronic Fatigue Syndrome patients from Italy: new data. J. Chronic Fatigue Syndr. 1996; 2:103-113.
12. Knox K, Brewer JH, Carrigan DR. Persistent active human herpesvirus 6 (HHV-6) infections in patients with chronic fatigue syndrome. J. Chronic Fatigue Syndr. 1999; 6:245-246.
13. Ablashi D, Eastman HB, Owen CB, Roman MM, et al. Frequent HHV-6 reactivation in multiple sclerosis (MS) and chronic fatigue syndrome (CFS) patients. J. Clin Virol. 2000; 16:179-191.
14. Wallace HL, Natelson B, Gruse W, Hay J. Human herpesviruses in chronic fatigue syndrome. Clin. Diagn. Lab. Immunol. 1999; 6:216-223.
15. Reeves WC, Stamey FR, Black JB, Mawie AC, Stewart JA, Pellett PE. Human herpesviruses 6 and 7 in chronic fatigue syndrome: a case control study. Clin. Infect. Dis. 2000; 31:48-52.
16. Martin WJ. Detection of RNA sequences in cultures of a stealth virus isolated from the cerebrospinal fluid of a health care worker with chronic fatigue syndrome. Case report. Pathobiology 1997; 65(1):57-60
17. Martin WJ. Simian cytomegalovirus-related stealth virus isolated from the cerebrospinal fluid of a patient with bipolar psychosis and acute encephalopathy. Pathobiology. 1996; 64(2):64-6.
18. Murdoch C, Finn A. Chemokine receptors and their role in inflammation and infectious diseases. Blood. 2000; 95:3032-43.
19. Nijs J, Nicolson GL, De Becker P, Coomans D, De Meirleir K. Prevalence of mycoplasmal infections among European chronic fatigue syndrome patients. . J. Chronic Fatigue Syndr. 2002; 10: In press.
20. Nicolson GL, Nasralla M, Nicolson NL, Haier J. High prevalence of mycoplasmal infections in symptomatic (Chronic Fatigue Syndrome) family members of mycoplasma-positive Gulf War Illness patients. J. Chronic Fatigue Syndr. 10: in press. [find article at www.immed.org]
21. Nicolson GL, Nicolson NL. Diagnosis and treatment of mycoplasmal infections in Persian Gulf War Illness-CFIDS patients. Intern. J. Occup. Med. Immunol. Tox. 1996; 5:69-78. [find article at www.immed.org]
22. Nicolson GL, Nicolson NL, Nasralla M. Mycoplasmal infections and Chronic Fatigue Illness (Gulf War Illness) associated with deployment to Operation Desert Storm. Intern. J. Med. 1988; 1:80-92. [find article at www.immed.org]
23. Thornton D. A survey of mycoplasma detection in vaccines. Vaccine 1986; 4:237-240.
24. Unwin C, Blatchley N, CokerW, et al. Health of UK servicemen who served in the Persian Gulf War. Lancet 1999; 353:169-178.
25. Nicolson GL, Nass M, Nicolson NL. The anthrax vaccine controversy. Questions about its efficacy, safety and strategy. Med. Sentinel 2000; 5:97-101. [find article at www.immed.org]
26. Nicolson GL, Nasralla M, Franco AR, et al. Diagnosis and Integrative Treatment of Intracellular Bacterial Infections in Chronic Fatigue and Fibromyalgia Syndromes, Gulf War Illness, Rheumatoid Arthritis and other Chronic Illnesses. Clin. Pract. Alt. Med. 2000; 1(2):92- 102. [find article at www.immed.org]
27. Nicolson GL. Considerations when undergoing treatment for chronic infections found in Chronic Fatigue Syndrome, Fibromyalgia Syndrome and Gulf War Illnesses. Part 1: Commentary. Part 2: Antibiotics and General Considerations. Intern. J. Med. 1998; 1:115-117 (Part 1), 1:123-128 (Part 2). [find article at www.immed.org]
28. Rea WJ, Pan Y, Johnson AR, Ross GH, et al. Reduction of chemical sensitivity by means of heat depuration, physical therapy and nutritional supplementation in a controlled environment. J. Nutrit. Environ. Med. 1996; 6: 141-148. For FMS, CFS/ME or RA or other autoimmune disease patients, The Institute for Molecular Medicine suggests the following lab tests (codes are IMD or CPT codes). Please note that patients should be off all antibiotics and immune enhancement products for 4 weeks prior to testing to insure that the test will not be a false-negative due to the antibiotic suppression of a blood infection. 1. Test Panel 1007 (CPT: 87798x3, 87581) Mycoplasma species panel of 4 pathogenic mycoplasmas (M. fermentans, M. penumoniae, M. hominis, M. penetrans) by PCR. Specimen Requirements: one (1) 5 cc Lavender top Plastic Tube (EDTA). The blood is collected, immediately mixed and placed on ice, then shipped on wet ice or immediately flash frozen and shipped with dry ice by courier (foreign shipments) to IMD to arrive within 24-36 hours. 2. Test 1006 (CPT: 87486) Chlamydia pneumoniae by PCR. Specimen Requirements: one (1) 5 cc Lavender top Plastic Tube (EDTA). Specimen Requirements: one (1) 5 cc Lavender top Plastic Tube (EDTA). The blood is collected, immediately mixed and placed on ice, then shipped on wet ice or immediately flash frozen and shipped with dry ice by courier to IMD to arrive within 24-36 hours. 3. Test 07056 (CPT: 87486) Brucella species by PCR. Justification: Specimen Requirements: one (1) 5 cc Lavender top Plastic Tube (EDTA). Specimen Requirements: one (1) 5 cc Lavender top Plastic Tube (EDTA). The blood is collected, immediately mixed and placed on ice, then shipped on wet ice or immediately flash frozen and shipped with dry ice by courier to IMD to arrive within 24-36 hours. 4. Test 07047 (CPT: 87476) Borrelia burgdorferi (Lyme Disease) by PCR. Specimen Requirements: one (1) 5 cc Lavender top Plastic Tube (EDTA). Specimen Requirements: one (1) 5 cc Lavender top Plastic Tube (EDTA). The blood is collected, immediately mixed and placed on ice, then shipped on wet ice or immediately flash frozen and shipped with dry ice by courier to IMD to arrive within 24-36 hours. 5. Test 07039 (CPT: 87532) Human herpes virus 6 (HHV-6) test by PCR. Specimen Requirements: Collect blood in one (1) 5 cc Lavender Top Plasma Tubes (EDTA), mixed and separate blood plasma by centrifugation. The plasma is then shipped on wet ice or immediately flash frozen and shipped with dry ice by courier to IMD to arrive within 24-36 hours. 6. Test 07034 (CPT: 87496) Cytomegalovirus (CMV) test by PCR. Specimen Requirements: Collect blood in one (1) 5 cc Lavender Top Plasma Tubes (EDTA), mixed and separate blood plasma by centrifugation. The plasma is then shipped on wet ice or immediately flash frozen and shipped with dry ice by courier to IMD to arrive within 24-36 hours.