Quantification of human cytomegalovirus DNA in peripheral blood polymorphonuclear leukocytes of immunocompromised patients by the polymerase chain reaction

Abstract

Human cytomegalovirus (HCMV) DNA amplification by the polymerase chain reaction (PCR) was utilized previously for successful monitoring of HCMV infections in immunocompromised patients. However, analysis of an extended series of clinical samples revealed the relatively frequent presence of PCR inhibitors. Hence, the need for availability of an internal control of the reaction allowing identification of false negative results. Similarly, an internal standard appeared necessary for quantification of viral DNA in clinical samples. For this purpose, we constructed a recombinant DNA molecule which could be amplified by the same set of primers used for HCMV DNA amplification. Coamplification of the recombinant DNA molecule and clinical samples proved to be a simple and reliable method for verifying sample competence for amplification. In addition, coamplification of serial known amounts of the same molecule, used as internal standard, and test sample, allowed quantification of viral DNA in polymorphonuclear leukocyte samples. Quantitative monitoring of HCMV infection and antiviral treatment may provide critical indications as to whether and when to initiate or discontinue antiviral treatment in immunocompromised patients with systemic HCMV infections.