Cytomegalovirus (CMV) is a major pathogen in immunosuppressed patients, especially in transplant recipients and in individuals with the acquired immunodeficiency syndrome (AIDS). The advent of effective antiviral chemotherapy for the treatment of various forms of CMV disease has placed a premium on the development of rapid and accurate diagnostic methods. Significant advances have been made employing monoclonal antibodies (mAb) to detect the virus with excellent sensitivity and specificity within 16-48 hours of specimen collection. However, methods which merely determine whether CMV is present or absent in various specimens cannot distinguish the patients with invasive CMV disease from those who shed the virus because of immunosuppression who have transiently positive CW blood leukocyte cultures. This is especially true of efforts to diagnose CMV pneumonitis through studies of bronchoalveolar lavage (BAL) fluids and to detect serious disseminated disease solely by demonstration of viremia. Diagnostic approaches which take into account the pathogenesis of CMV dissemination and disease at the molecular level are most likely to be useful in this regard if they can be adapted for use in modern diagnostic virology laboratories. In this proposal, we present evidence employing quantitative DNA blot hybridization that high levels (100 pg of viral DNA or greater per 10 ug leukocyte DNA) of circulating CMV DNA in blood leukocytes are closely correlated with the presence of visceral organ CMV disease in solid organ transplant recipients and in patients with AIDS. This is not the case, however, in marrow recipients. Our preliminary studies also suggest that large amounts of CMV DNA in BAL cells are correlated with the presence of CMV pneumonitis verified histologically. We now propose to test these observations further and to develop non-radioisotopic assays for quantitation of CMV DNA in these samples. In addition, we will determine whether CMV visceral organ disease and interstitial pneumonitis are correlated with the replicative state of CMV within peripheral blood leukocytes and BAL cells, respectively. For these studies, we will employ extremely sensitive single strand RNA probes which hybridize to specific CMV messenger RNAs and mAbs which recognize defined protein gene products. In these studies, it will also bc determined whether the number of infected blood leukocytes and BAL cells is associated with visceral organ involvement and pneumonitis, respectively. As we identify quantitative or qualitative molecular correlates of CMV CL disease which would be useful in diagnosis, we will develop non-radioisotopic assay systems suitable for use in diagnostic virology laboratories. Because of the diverse and large group of patients with CMV disease at our institution, these studies can be conducted expeditiously and will result in the development of diagnostic assays which identify the patients in need of antiviral chemotherapy.
