Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2002-02-13
2004-09-14
Housel, James (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024320, C536S023100, C536S023720, C435S004000, C435S005000, C435S006120, C435S091200
Reexamination Certificate
active
06790952
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates generally to novel compositions and methods for detecting the presence of viruses that frequently infect humans and are associated with the development of human disease. More particularly, the invention is directed to an accurate and sensitive method for the diagnosis and quantitation of Epstein-Barr virus infection using specific oligonucleotides as primers to amplify particular regions of the genome of the virus sought to be detected in a clinical specimen. Epstein-Barr virus-specific oligonucleotides may be used in the subsequent detection of the amplified regions of DNA.
Epstein-Barr virus (EBV), a human herpes virus, is ubiquitous in humans. Antibodies to polypeptides of the virus are present in over 80% of human serum samples from the United States and in even higher percentages from populations in Asia and Africa. Although it is prevalent throughout the world, the consequences of EBV infection vary among different populations. The virus is responsible for of infectious mononucleosis, a benign proliferation of infected B-lymphocytes, in Western countries and is implicated in Burkitt's lymphoma in Africa and nasopharyngeal carcinoma (NPC) in Asia. EBV can also cause acute and rapidly progressive B lymphoproliferative disease in severely immune compromised patients.
As a result of the immunosuppression necessary to maintain the function of transplanted organs, transplant patients are all at risk for developing EBV infection and therefore post transplant lymphoproliferative disorder (PTLD). However, the group at highest risk for this complication is the liver transplant population. This is because these patients are generally very young, frequently less than 5 years of age, and therefore they frequently have not yet been exposed to EBV and as a result do not have a natural immunity to the virus.
Approximately 50% of the transplanted population develop EBV infection during the post-transplant course, frequently symptomatic including PTLD. These patients therefore are a prime population for monitoring the development of EBV infection and its associated complications. Approximately 50% of these patients will develop EBV infection requiring monitoring of the peripheral blood EBV DNA levels.
Nationally there are between 450-500 liver transplants in pediatric patients. In those patients under 14 months of age approximately 75% of them develop EBV infections. These numbers do not include all the bone marrow, kidney, heart, and lung transplants in pediatric patients. And although there is a lower incidence of EBV lymphoproliferative complication in all of the adult transplant patients, there is a clinically significant incidence of this complication in these patients as well. The demand for this kind of patient monitoring therefore is high. Currently, there are a few recognized labs who provide quantitative EBV analyses.
EBV associated post-transplant lymphoproliferative disease (PTLD) is a major cause of morbidity and mortality for children and adults who undergo solid organ transplantation and bone marrow transplantation (Ho M, et al. The frequency of Epstein-Barr virus infection and associated lymphoproliferative syndrome after transplantation and its manifestations in children. Transplantation 45:719-727, 1988).
Patients who are EBV naïve and receive an organ from an EBV positive donor, especially those treated with anti-T-cell immunotherapy (anti-thymocyte globulin or monoclonal antibodies), are at high risk for developing PTLD. Infants and toddlers, who comprise 50% of the pediatric liver transplant population, are usually EBV naïve. More than 75% of patients at risk acquire the virus within the first year of life. Up to 15% of liver transplant recipients who are at high risk will develop PTLD. Consequently, the risk of infection and complication is substantial.
For children, especially those less than two years of age, PTLD is a critical factor affecting cost, graft function and quality of life. Attempts to treat these transplant patients with interferon, acyclovir, and anti-B cell monoclonal antibodies are generally not successful once an EBV infection has been established or reactivated (Papadopoulos E B, et al. Infusions of donor leukocytes as treatment of Epstein-Barr virus associated lymphoproliferative disorder complicating allogeneic marrow transplantation. N Engl J Med 330: 1185, 1994; Zutter M M, et al. Epstein-Barr virus lymphoproliferation after bone marrow transplantation. Blood 72, 520, 1988; Shapiro R S, et al. Epstein-Barr virus associated B-cell lymphoproliferative disorders following bone marrow transplantation. Blood 71: 1234, 1988; Antin J et al. Selective depletion of bone marrow T lymphocytes with anti-CD5 monoclonal antibodies: Effective prophylaxis for graft-vs-host disease in patients with hematologic malignancies. Blood: 78: 2139, 1991; Martin P J, et al. Fatal Epstein-Barr virus associated proliferation of donor B cells after treatment of acute graft vs-host disease with a murine anti-T-cell antibody. Ann Intern Med 101:310, 1984). A number of studies however have shown that PTLD may be reversible in solid-organ transplant recipients following reduction or discontinuation of immune suppression (Starzl T, et al. Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporin steroid therapy. Lancet I: 583, 1984).
Differences in levels of EBV DNA found in the peripheral blood post-transplant may distinguish which patients are at highest risk to develop EBV PTLD and therefore could permit earlier intervention in these patients. Previous studies have shown a correlation between levels of EBV DNA and the occurrence of EBV-PLTD in organ transplant patients (Kenagy D N, et al. Epstein-Barr virus DNA in peripheral blood leukocytes with post-transplant lymphoproliferative disease. Transplant 60: 547, 1995; Riddler S A, et al. Increased levels of circulating Epstein-Barr virus (EBV) infected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of post-transplant lymphoproliferative disease in solid-organ transplant recipients Blood 84: 972, 1994; Savoie A, et al. Direct correlation between the load of Epstein-Barr virus infected lymphocytes in the peripheral blood of pediatric transplant patients and risk of lymphoproliferative disease. Blood 83: 2715, 1994). These results suggest the potential benefit of monitoring EBV DNA levels in the peripheral blood of the transplant patients may be clinically useful in the management and possible preemptive intervention of the development of PTLD in these patients. As a result, a number of assays using a variety of approaches have been developed and reported for either semiquantitative or quantitative determination of EBV DNA levels.
Most of the semiquantitative assays are based on standard PCR analysis of a dilution series of patient samples compared to a known standard. While extremely useful, quantitative PCR can be very laborious to perform. Most of the difficulties arise because only a very small number of the cycles in a PCR reaction contain useful information. The early cycles have undetectable amounts of the DNA product and late cycles (plateau phase) are almost as uninformative. The PCR product is then detected by gel electrophoresis followed by ethidium bromide staining of the gel. The result is determined by the lowest dilution at which a band is visible (Lucas K G, et al. Semiquantitative Epstein-Barr virus polymerase chain reaction for the determination of patients at risk for EBV induced lymphoproliferative disease after stem cell transplantation. Blood 91: 3654, 1998; Baldanti F, et al. High levels of Epstein-Barr virus DNA in blood of solid organ transplant recipients and their value in predicting post-transplant lymphoproliferative disorders. J. Clin Microbiol. 38: 613, 2000; Rogers B, et al. Epstein-Barr virus polymerase chain reaction and serology in pediatric post-transplant lymphoproliferative disorder: three year experience. Pediatric & Developmental Pathology 1: 480, 1998
Groen Pamela A.
Witte David P.
Cincinnati Children's Hospital Research Foundation
Frost Brown Todd LLC
Housel James
Li Baoqun
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