Nucleic Acid Sequence-Based Amplification: A New Technique for Monitoring Cytomegalovirus Infection in Transplant Recipients M.J. Blok, I. Lautenschlager, M.H.L. Christiaans, J.P. Van Hooff, V.J. Goossens, J.M. Middeldorp, P. Sillekens, A. Ramon, K. Ho ¨ ckerstedt, and C.A. Bruggeman T HE MAJORITY of the adult human population is infected with cytomegalovirus (CMV), a member of the -herpesvirus family. Normally, infection of immuno- competent individuals does not cause disease. In contrast, infection of immunocompromised individuals, such as neo- nates, patients with AIDS, and transplant recipients, can cause severe complications and can even lead to death. Therefore, early detection of active CMV infection is necessary to start effective antiviral treatment. Nucleic acid sequence-based amplification (NASBA) has been devel- oped for the specific amplification of RNA. 1 We evaluated the diagnostic value of monitoring the expression of the CMV immediate early (IE) 1 mRNA using NASBA, in peripheral blood cells of kidney and liver transplant pa- tients. The IE 1 mRNA is expressed directly after entrance of the virus into a cell. NASBA results were compared to the techniques which are routinely being used for the detection of CMV: pp65-antigenemia, virus culture, and serology. MATERIALS AND METHODS Patients A total of 489 and 178 whole blood samples were prospectively collected from 42 kidney and 23 liver transplant recipients, respec- tively. The patients were grouped according to the CMV serostatus of the kidney donor (D) and the recipient (R), resulting in the following distribution for kidney transplant patients: 13 D+/R+;8 D+/R-; 13 D-/R+;8D-/R-, and for liver transplant patients: 12 D+/R+;2D+/R-;5D-/R+;2D-/R- (for two additional patients the serostatus of the donor or the recipient was not known). Definition of CMV Infection CMV infection was defined by at least two positive results for one or more of the following items: virus culture, antigenemia, presence of anti-CMV-IgM and/or a significant rise (at least four times, compared to the pre-transplantation titer) of anti-CMV-IgG levels. Definition of CMV Disease According to the criteria of Metselaar, 2 consisting of fever for at least 3 consecutive days, leukocytopenia, thrombocytopenia, liver abnormalities and organ involvement, confirmed by concomitant positive virus culture and/or antigenemia results. Virus Culture Leukocytes isolated from whole blood samples were cocultured with human embryonic fibroblasts (HEF). The virus cultures were stained after 2 days with MAbs directed against the IE 1 protein (detection of early antigen fluorescent foci [DEAFF]). 3 In addition, virus cultures were maintained for a period of 6 weeks and observed for the cytopathic effect (CPE) of replicating virus. DEAFF and CPE results were combined to get one outcome for detection of virus by cell culture. pp65 Antigenemia The pp65 antigen, a major early protein, was detected immunocy- tochemically in leukocytes isolated from whole blood samples, using antibodies directed against pp65. This assay was performed essentially as described by Van der Bij et al. 4 Serology The ImXCMV and AXSym assays (Abbott Laboratories) were respectively used for the detection of CMV-specific IgM and IgG antibodies. NASBA Nucleic acids were isolated from whole blood samples using the Boom method. 5 Briefly, 100 L heparinized whole blood was added to 900 L NASBA lysis buffer, containing guanidium thiocyanate. The nucleic acids were subsequently bound to silica and washed with ethanol and acetone to remove residual cell debris. The nucleic acids were eluted from the silica after drying it. The isothermal (41°C) NASBA amplification reaction is based on the action of three different enzymes: avian myeloblastosis reverse transcriptase, RNaseH, and T7 RNA polymerase. This amplifica- tion procedure can reach a 10 12 -fold amplification of the target RNA, with a sensitivity of approximately 10 copies. A primer set was used which anneals to sequences in exon 4 of the IE 1 mRNA. The detection of the amplification products is based on electro- chemiluminescence (ECL). From the Departments of Medical Microbiology, and Internal Medicine, University Hospital Maastricht, Maastricht, The Neth- erlands; the Department of Virology and Transplantation Unit, Department of Surgery, University Hospital Helsinki, Helsinki, Finland; and Organon Teknika B.V., Boxtel, The Netherlands. Address reprint requests to M.J. Blok, Department of Medical Microbiology, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands. 0041-1345/99/$–see front matter © 1999 by Elsevier Science Inc. PII S0041-1345(98)01639-X 655 Avenue of the Americas, New York, NY 10010 308 Transplantation Proceedings, 31, 308–309 (1999)