DISCORDANT DETECTION OF HUMAN CYTOMEGALOVIRUS DNA FROM PERIPHERAL-BLOOD MONONUCLEAR-CELLS, GRANULOCYTES AND PLASMA - CORRELATION TO VIREMIAAND HCMV INFECTION

Background: There exist only few data about the HCMV infection of sing le positive leukocyte subtypes in immunosuppressed patients. Most repo rts describe HCMV coinfection of cells of the myelomonocytic line or e ven T- and B-cell populations. Correlation of positive PCR findings fr om two major leukocyte fractions and plasma to viremia and HCMV infect ion in general should contribute to select suitable sources of HCMV DN A for diagnostic purposes. Objective: The diagnostic value of qualitat ive leukoDNAemia of simultaneously isolated peripheral blood mononucle ar cells (PBMC), granulocytes as well as plasmaDNAemia was evaluated b y comparing the positive results of nested PCR from blood with virus i solation either from leukocytes or from any other sources, with serolo gy and the clinical status of immunosuppressed patients. Study design: PBMC, granulocytes and plasma were prepared of a total of 220 blood s amples of 75 immunosuppressed patients with clinically suspected prima ry or recurrent HCMV infection. In a collective of 35 patients consist ing mainly of recipients of marrow or solid organ transplants positive results of leuko- or plasmaDNAemia were correlated with data from HCM V screening and the clinical status. For standardization, HCMV IE Exon 4 DNA was amplified from 100 ng cellular DNA of each leukocyte popula tion. Cross contamination can be excluded. DNA from plasma was extract ed by phenol/chloroform. Using this experimental design, HCMV DNA was not detectable in PBMC, granulocytes and plasma of 23 healthy HCMV ser opositive blood donors. Results: Leukocyte separation in a collective of 30 patients with positive leukoDNAemia revealed in only 12 cases (4 0%) double infection of PBMC and granulocytes. In the majority of case s (18 patients, 60%) however, HCMV DNA was detectable in only one leuk ocyte fraction, either in PBMC or granulocytes. LeukoDNAemia did not c orrelate to viremia. HCMV DNA. amplified from plasma was shown to be c ell free. infectious virus from plasma was not isolated. The predictiv e value of qualitative nested PCR from blood to detect HCMV infection was high for plasma and decreased in the following sequence: plasma (0 .92) > PBMC (0.83) > granulocytes (0.65). Conclusions: Qualitative nPC R from plasma and PBMC seems to be sufficient to detect (an ongoing) H CMV infection of immunosuppressed patients. However, the rate of singl e positive leukocyte fractions is similar to 60%. Therefore, viral leu koDNAemia in 40% of cases seems to be restricted to either PBMC or gra nulocytes. For diagnostic purposes the whole leukocyte population shou ld be used for PCR analysis. (C) 1998 Elsevier Science B.V. All rights reserved.