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Grant: Development of a Variant-specific, Calibrated Real-time PCR Assay for the Absolute Quantification of HHV-6A and B Nucleic Acids in Clinical Samples PI: Mauro S. Malnati, MD, San Raffaele Scientific Institute, Milan, Italy USA While several diagnostic procedures have been developed that distinguish between the HHV-6A and B variants, there is still no highly sensitive and reliable procedure that can determine the concentration of HHV-6 in clinical specimens for the absolute quantification of the HHV-6A and HHV-6B DNA load in different types of clinical samples. The development of such an assay is particularly challenging in the case of mixed infections with HHV6-A and B, since the two variants can interfere with each other, leading to false negative results. The availability of a highly sensitive and accurate assay is essential for conducting clinical studies to elucidate the possible pathogenic role of the two viral variants in a variety of diseases. The assay will also aid in assessing which variant is involved with which disease. Dr. Mauro Malnati, the Deputy Chief of Human Virology at the DIBIT – S. Raffaele Scientific Institute, is designing a calibrated Real-Time PCR assay based on the combination of TaqMan PCR and “calibrator” technologies. The TaqMan technology is the most reliable and widely tested technology developed for Real-Time PCR as it displays an outstanding degree of specificity, accuracy, as well as cost and time efficiency. The “calibrator” technology, developed and patented in Dr. Malnati’s laboratory, is standardized for the detection of false-negative results and provides absolute quantification. Because of the improved accuracy of quantification, it also decreases the degree of inter-sample variability. Finally, the calibrator technology helps to determine a cut-off value sensitivity for each negative sample which would provide clinicians with a more accurate interpretation of the negative result and thus a more complete patient evaluation. Dr. Malnati intends to test various HHV-6A and B isolates to see that results are consistent. The isolates will be provided through the Foundation repository. We are in the process of obtaining fresh HHV-6B isolates from Dr. Yoshikawa and three HHV-6A isolates are already in the repository which Dr. Ablashi had when he was at NCI. We also have another HHV-6 isolate from a CFS patient which seems to have properties of both A & B variants (DA isolate) that will also be sent to Dr. Malnati as well as Dr. Tang for their variant specific PCR assays.
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