Quantitative Real-Time Polymerase Chain Reaction for Evaluating DNAemia due to Cytomegalovirus, Epstein-Barr Virus, and BK Virus in Solid-Organ Transplant Recipients

Abstract

Testing for cytomegalovirus-, Epstein-Barr virus-, and BK virus-specific gene targets in specimens from solid-organ transplant recipients for DNA by quantitative real-time polymerase chain reaction has been implemented in many diagnostic facilities. This technology provides rapid, accurate, and reproducible results for early detection, monitoring, and medical management of patients with these infections. Because these assays are becoming commonly used in clinical practice, the technical variables associated with specimen processing (e.g., nucleic acid extraction, gene target, and result reporting), amplification, and unique patient characteristics (e.g., age, sex, underlying diseases, immune status, and immunosuppressive regimens received) are factors that may influence the understanding and interpretation of test results. We emphasize the need for standardization of existing variables through parallel comparative and proficiency testing, uniform units for expressing results, to provide for clinical correlation with the results of these molecular assays.Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK virus (BKV) are common pathogens and major causes of morbidity and mortality in patients who have received a solid-organ transplant [1][2][3]. Infection with these viruses is indicated by demonstrating the presence of the virus in tissue and/or blood specimens. Because all of these viruses (i.e., CMV, EBV, and BKV) contain DNA, the term "DNAemia" certainly applies. The more general "viremia" can apply to a bloodstream infection caused by a DNA-or RNA-containing virus. BKV and EBV are not detected in cell cultures by routine diagnostic virology methods. Detection of CMV in blood specimens can be qualitatively recognized rapidly (16 h after inoculation) in shell vial cultures, but quantitation of the CMV load with this technology is cumbersome and impractical.The first laboratory method for quantitation of CMV load was the antigenemia test [4] jective interpretation of the test results [5][6][7]. Importantly, both shell vial cell cultures (qualitative) and antigenemia tests (quantitative) have been shown in comparative studies to be less sensitive than PCR. In addition, DNAemia was detected in an earlier stage of nucleic acid amplification [6][7][8][9][10].As an alternative to cell culture and the antigenemia test, implementation of molecular techniques for the rapid and sensitive detection of nucleic acid targets has yielded new levels of capabilities for laboratory diagnosis of many microbial infections [11]. Implementation of these tests has been facilitated by the availability of real-time PCR instrumentation with automation of nucleic acid-target amplification and amplicondetection steps in a "closed system" (i.e., reaction tubes never opened during or after amplification). Therefore, for both qualitative and quantitative detection of viruses, automated realtime PCR has generally replaced the time-consuming and laborintensive conventional amplification and detection of products by gel electrophoresis and ...

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