Different expression of human herpesvirus-6 (HHV-6) load in whole blood may have a significant impact on the diagnosis of active infection.

BACKGROUND: Viral load in whole blood is the main virological marker for assessing HHV-6 infection and is used as an indication to begin antiviral therapy. Results are usually expressed as the number of genomic equivalent copies (gec) per mL of blood, although HHV-6 DNA in blood is mainly localized in lymphocytes and polymorphonuclear leukocytes. OBJECTIVES: Since leukocyte counts vary in immunocompromised patients, especially in stem cell transplant recipients, the aim of this study was to compare HHV-6 load expressed as gec per mL with load expressed as gec per million cells (mc), using quantitative real-time PCR for HHV-6 and cell DNA. STUDY DESIGN: 194 blood samples from 101 patients were analyzed. Leukocyte count was obtained for 142 samples. RESULTS: The two modes of expression were incompletely correlated (p<0.0001; R(2)=0.732). To understand this relative discrepancy, samples were classified according to hematological criteria (normal leukocyte count, leukopenia, agranulocytosis, lymphopenia). The expression modes were correlated in all cases except for agranulocytosis (p=0.21; R(2)=0.087). Moreover, the median of ratio between gec per mc and gec per mL ranged from 0.5 when leukocyte count was normal, to 8.2 in cases of agranulocytosis. HHV-6 load follow-up suggested that in agranulocytosis expressing results as gec per mc tended to provide a more representative result. CONCLUSIONS: The different expression of HHV-6 load in whole blood, as either gec per mL or gec per mc resulted in different estimations of infection in the case of agranulocytosis. In this situation, the latter mode of expression is preferred.

Multicenter quality control for the detection of hepatitis C virus RNA in seminal plasma specimens.

The discrepant results available in the literature about the presence of hepatitis C virus (HCV) RNA in seminal plasma of men chronically infected by this agent are related, at least in part, to the molecular techniques used and particularly to the wide range of protocols dedicated to RNA extraction. In order to evaluate these protocols and to standardize the method of detection of HCV RNA in this fluid, a panel of coded specimens was tested blindly in 12 French laboratories; it included 14 seminal plasma specimens and four water controls spiked with HCV RNA ranging from 10 to 20000 IU/ml and two HCV-negative seminal plasma specimens. The extraction step was performed according to methods using either silica beads (NucliSens [Organon Teknika S.A., Fresnes, France]; RNA viral kit [Qiagen, Courtaboeuf, France]) or guanidinium thiocyanate (Amplicor HCV assay; Roche Diagnostics, Meylan, France), preceded or not by a centrifugation of the seminal plasma. For the amplification step, all the laboratories performed the same reverse transcription-PCR technique (Amplicor HCV Cobas assay). The percentage of correct results ranged from 53.3 to 100, the poorest results being obtained when no centrifugation step preceded the Amplicor extraction protocol. The rate of correct results was significantly higher in laboratories using a preliminary centrifugation of the specimen (P = 0.034 by chi-square test). By contrast, the overall number of correct results was not correlated to the initial volume of sample used for the test. These results allowed us to validate standardized techniques adapted to the performance of this test on a routine basis, especially in men infected with HCV and involved in programs of medically assisted reproduction.