Herpes Virus Testing
Epstein-Barr Virus (EBV/HHV-4)
Detection of Epstein-Barr Virus: A Rapid Screening Test to Aid in The Diagnosis of Chronic Fatigue Syndrome
Discovered in the 1950’s by Denis Burkitt as an unrecognized cancer affecting children in Africa (Burkitt’s Lymphoma), HHV-4 was later identified as a herpes virus by Tony Epstein and Yvonne Barr, for whom the virus was named. Since its discovery Epstein-Barr virus (EBV) has been found to be extremely prevalent in the human population affecting >95% of the world’s population by early adulthood.
What is HHV-4?
Part of the lymphocryptovirus genus of gammaherpesviridae, Epstein-Barr Virus shares atropism for B lymphocytes, and epithelial cells. EBV targets primary B lymphocytes, with a latent infection, and triggers sustained cell proliferation.
Virus morphology is identical to that of other members of the herpes virus family. EBV is a 172kb double stranded linear DNA molecule encoding some 80-100 genes. Upon infection of a B lymphocyte the DNA becomes circular, and forms independent episomes via recombination of homologous terminal repeat sequences.
In latent cell lines studied in vitro, EBV gene expression appears to be limited to about nine of the genes encoded by the virus. Six of these encode the nuclear antigen (EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and LP) while three encode for the membrane proteins (LMP-1, LMP-2A and LMP-2B). Remaining transcription appears to be non-polyadenylated RNA associated with interferon induced pathways.
Epidemiology
Since it discovery Epstein-Barr virus (EBV) has been found to be extremely prevalent in the human population affecting about 50% of U.S. children by age 5, with >95% of the world's population infected by early adulthood. Known as infectious mononucleosis (kissing disease), infection of adolescent populations is quite large due to the exchange of saliva. The typical incubation period is between 30-50 days with viral DNA persistent at high levels in the saliva of patients as long as six months after initial infection, continuing intermittently through life. EBV, like herpes simplex 1 and 2, can be transmitted by blood transfusion or tissue transplantation.
What are the Symptoms and Diagnosis?
The usual outcome of infection is B-cell activation and benign proliferation which may produce infectious mononucleosis; however HHV-4 infection can become complicated in immuno-compromised patients. EBV should be suspected if a patient has symptoms of infectious mononucleosis.
EBV and Chronic Fatigue Syndrome
Once thought to be the cause of chronic fatigue syndrome (CFS), EBV has been determined to play no part in the onset of CFS. Clinical research has determined that the presence of EBV in CFS patients is more likely to be a secondary viral reactivation.
Epstein-Barr Virus (EBVD) Test
Background:
Nested-PCR targets the episomal EBV and detects both Type 1 and Type 2 viruses of EBV equally as
assessed using a racemic primer mixture. The product of the outer primers in this system is 407 base pairs
in length while the product of the inner primers is 293 base pairs long. This PCR system has been
successfully used to detect EBV DNA in the sera of patients with chronic fatigue syndrome.
Method:
Nested-PCR
Result:
Qualitative only. The results for this analysis are presented as positive or negative for the presence of EBV.
Results and Interpretation
Results are qualitative, positive/negative, with normal reference range as negative. The results of this test should be used in addition to all other relevant clinical data before making a diagnosis and/or a recommendation for treatment. Ranges are subject to change dependent on future data. This test was developed and its performance characteristics verified by REDLABS U.S.A.
Human Cytomegalovirus (HCMV/HHV-5)
Detection Of Cytomegalovirus:
A Rapid Screening Test to Aid in the Diagnosis of Chronic Fatigue Syndrome
Cytomegalovirus (CMV) is found universally throughout the world and by age 40 between 50% and 80% of adults in the United States have been infected. Most cases, however, are asymptomatic for CMV infection with no long-term consequences. CMV can cause complications for immuno-suppressed persons, such as HIV infected patients, transplant patients, and newborn infants exposed in utero.
What is Hhv-5?
Cytomegalovirus (CMV) is part of the ß-Herpesviridae family, and is the largest of the herpes viruses. The complete genome sequence, some 230kbp, is known and has been studied in great detail. Upon infection the DNA becomes circular and forms independent episomes via recombination of homologous terminal repeat sequences.
HHV-5 DNA appears to be segregated to the monocytes in healthy carriers with very little viral DNA detectable in the T or B cells. Analysis by PCR suggests the frequency of infected cell in a PBMC preparation may be as little as 1 in 10,000/ preparation. Clinical studies suggest that the virus is in the peripheral blood compartment. Bone marrow, the site of lymphocyte production, appears to act as a reservoir for HHV-5, but the progenitor cells of monocytes (CD34+ stem cells) have little or no viral gene expression in vivo. Using leukocyte depleted blood products reduces the risk of transmission; however, healthy HHV-5 positive blood donors can transmit infection to recipients.
Epidemiology
Infections with CMV are common and reach most of the population. Although HHV-5 is the recognized cause of CMV mononucleosis, symptoms of HHV-5 infection are rare in immuno-competent people. The eminent danger of CMV infection is within immuno-compromised groups such as immature neonates, organ transplant recipients and HIV infected individuals. In this population CMV can cause severe disease.
What Are The Symptoms And Diagnosis?
Most people infected with CMV manifest no symptomology, even during periods of viral reactivation. When symptoms manifest some experience a mononucleosis-like syndrome with prolonged fever and mild hepatitis. In all infected people, the virus remains for life; however, in the majority of cases the virus is dormant and recurrence is low unless the immune system becomes suppressed due to therapeutic drugs or disease.
CMV should be suspected if a patient:
- has symptoms of infectious mononucleosis but has negative test results for mononucleosis and Epstein Barr Virus (HHV-4), or,
- shows signs of hepatitis, but has negative test results for hepatitis A, B, and C.
Laboratory tests for active and latent CMV infection exist, though latent infection is difficult to isolate due to the low occurrence of infected cells in vivo. For best diagnostic results of latent infection, laboratory tests for CMV viral DNA by nested PCR performed by using peripheral mononuclear blood cell (PBMC) preparations. For active viral infection paired blood and serum or blood and urine specimens should be collected to RT-PCR analysis. One blood or serum sample should be taken upon suspicion of CMV, and another one taken within 2 weeks for vial load comparison.
Fetal infection with CMV can be serious, and can result in hearing loss, vision impairment, and varying degrees of mental retardation. The risk to the fetus occurs when the mother experiences primary, first time infection, during or six months immediately prior to pregnancy. However, routine laboratory testing for CMV of all pregnant women or women planning on becoming pregnant is cost prohibitive and should be evaluated on a case-by-case basis.
Cytomegalovirus (CMVD)
Background:
Nested-PCR targets the episomal CMV and detects the virus DNA of CMV as assessed using a primer.
The product of the outer primers in this system is 500 base pairs in length while the product of the
inner primers is 250 base pairs long. This PCR system has been successfully used to detect CMV DNA in
patients with both active and latent viral infection.
Method:
Nested-PCR
Result:
Qualitative only. The results for this analysis are presented as positive or negative for the presence of CMV.
Results and Interpretation
Results are qualitative, positive/negative, with normal reference range as negative. The results of this test should be used in addition to all other relevant clinical data before making a diagnosis and/or a recommendation for treatment. Ranges are subject to change dependent on future data. This test was developed and its performance characteristics verified by REDLABS U.S.A.
Human Herpesvirus Type 6
A number of recent publications have demonstrated a statistical association between persistent infection with Human Herpesvirus Type VI (HHV-6) and a number of chronic immune diseases (e.g., multiple sclerosis, B-cell lymphoma). Since persistent viral infection can cause over-induction of the interferon inducible enzymes – one of the biochemical hallmarks in CFS patients- detection of HHV-6 infection may be important as a biological marker of active autoimmune disease. There are two distinct subtypes of HHV-6: A and B variants. Type A is associated with more severe disease while Type B occurs within the general population often without associated morbidity.
While HHV-6-B roseola infections are easily detected, HHV-6-A detection is more difficult. The “A” variant can remain active in brain tissue, often with no trace in the serum.
One reason that it is difficult to detect HHV-6 is that since over 95% of us have been exposed to HHV-6-B in the past and since HHV-6 remains latent in cells, tests must be done serum (the clear liquid portion of the blood) to differentiate active from latent infection. In most viral infections, large numbers of virions spill into the plasma when the virus is replicating but this is not the case with HHV-6 because it is spread largely cell-to-cell or directly through the cells’ walls. This means that very little of it ends up in the serum so PCR tests must be extremely sensitive to persistent HHV-6 infections, once the acute phase is over. The “A” variant is 13% different in genome from the “B” variant and is more aggressive and lytic to the cells it infects.
Results and Interpretation
Results are qualitative. If the specimen is "positive" for HHV-6, the subtype will automatically be determined and reported at no extra charge.
Human Herpesvirus Type 7
HHV-7 is not associated with any one disease, although increased prevalence has been noted in patients with autoimmune disease and in children with exanthema subitum (roseola infantum), a mild virus disease of infants and children that is characterized by fever lasting three days followed by an eruption of rose-colored spots.
Results and Interpretation
Results are qualitative. If present, the result will be reported as “positive” for the presence of the organism.
Human Herpesvirus Type 8
HHV-8 is most typically identified with Kaposi’s sarcoma, the virus being present in the plaques or skin lesions. The virus has also been associated with HIV as being a possible co-factor of disease progression.
Results and Interpretation
Results are qualitative. If present, the result will be reported as “positive” for the presence of the organism.
Mycoplasma Species Detection by PCR
Mycoplasma are a group of the smallest organisms known and are associated with a variety of pathologies in a broad range of hosts. Infection with Mycoplasma can lead directly to pneumonia, arthritis, mastitis and urogenital disease. They have been detected in synovial fluid from patients with rheumatoid arthritis. In addition, certain Mycoplasma species have been demonstrated to be co-factors in the progression HIV-1 infection to AIDS by inhibiting cell proliferation and inducing programmed cell death (apoptosis) and concomitant fragmentation of cellular DNA. A number of recent publications in the medical literature have reported an increased incidence of Mycoplasma infection in patients with CFS.
Results and Interpretation
Results are qualitative. If present, the result will be reported as "positive" for the presence of the organism. In addition, speciation will be performed and provided with the result.
Chlamydia Pneumoniae Detection by PCR
C. pneumoniae is an opportunistic pathogen in humans, most often causing respiratory complications in immuno-compromised patients. Recent literature has linked infections with C. pneumoniae to chronic immune diseases such as Multiple Sclerosis. Further, the presence of Chlamydial antigens have been linked to coronary artery disease in that the immune response to these antigens cross-reacts with normal cell surface proteins, causing an inflammatory reaction and subsequent pathology.
Results and Interpretation
Results are qualitative. If present, the result will be reported as "positive" for the presence of the organism.
For more information on HHV-6 please visit the HHV-6 Foundation website at www.hhv-6foundation.org.