THREE CMV pp65 ANTIGENEMIA ASSAYS VERSUS QUANTITATIVE REAL TIME PCR AN UNJUST COMPARISON

Background: Cytomegalovirus (CMV), a member of the Human Herpesviruses, continues to be a significant cause of morbidity and mortality, especially in immunocompromised patients and transplant recipients. CMV-disease can be prevented by early detection and constant monitoring of high risk patients. Hence, quantitative assays for CMV such as Antigenemia and Real Time polymerase chain reaction (RTPCR) are currently the methods of choice. In this study we describe the comparison of three pp65 Antigenemia assays with a pp65 RTPCR.

Methods: From February through July 2007, a total of 137 whole blood EDTA samples (30 % pediatric, 92% transplant recipients) were subject to parallel testing on three different pp65Antigenemia assays, the CMV pp65 IFA (Chemicon, Light Diagnostics^TM), the CMV Brite^TM Turbo kit (Biotest Diagnostics Corporation), and an altered and validated in-house assay using the Chemicon kit. For each assay a minimum of 1 ml of specimen was adjusted to a white blood cell count of 1x10⁶/ml and processed according to the respective protocols. All samples were resulted as “number of positive WBC cells per 200,000” and evaluated for fluorescent stain intensity. Ninety-seven of the samples were aliquoted (220µL) and underwent DNA extraction and subsequent RTPCR targeting the pp65 gene for comparison.

Results: The best performance was achieved by the in-house Antigenemia assay (Chemicon) detecting the most positive samples (n=15, 10.9%) and yielding the highest intensity of fluorescence stain. The unaltered CMV pp65 IFA kit (Chemicon) detected 13 positive specimens (9.5%) with slightly lower stain intensities. CMV Brite^TM Turbo kit detected nine positive samples (6.6%), had the lowest average positive cell number and the lowest fluorescent stain intensity. Of the 97 specimens tested in parallel with RTPCR, all positive antigenemia results were confirmed by RTPCR, and the quantitated PCR values correlated with the antigenemia results. However, seven more low positive samples were detected by RTPCR (limit of detection: 100 copies/ml).

Conclusions: The CMV pp65 IFA kit was the most sensitive Antigenemia assay. Altering the protocol during specimen processing, WBC separation and RBC lysing, can enhance the performance of this kit significantly. In addition, the stronger fluorescent intensity noted for the positives cells allows for a less subjective reading. Overall, RTPCR was the most reliable, sensitive and reproducible method for CMV detection.