Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR

Abstract

Background: Human herpesvirus 6 (HHV-6) latently infects a majority of adults. In about 1% of the population HHV-6 exists in a chromosomally integrated form (ciHHV-6) that resides in every somatic and germ cell and can be transmitted through the germ line. Patients with ciHHV-6 have been misdiagnosed and unnecessarily treated for active HHV-6 infection, sometimes with important side effects, based on results from quantitative molecular HHV-6 tests.

Methods: A droplet digital PCR (ddPCR) assay was developed to identify ciHHV-6 in cellular patient samples by precisely determining the ratio of HHV-6 to cellular DNA. We validated the assay on confirmed ciHHV-6 patient samples and a cell line derived from a ciHHV-6 patient, and we analyzed hematopoietic stem cell transplant patients suspected of having ciHHV-6. We additionally evaluated whether the assay could be applied to stored plasma samples from a study of clinical correlates of HHV-6.

Results: The ddPCR assay accurately identified ciHHV-6 in cellular samples (buffy coat, peripheral blood mononuclear cells), giving a ratio very close to 1 HHV-6/cell [mean (SD), 1.02 (0.03)] in fluorescence in situ hybridization-confirmed sample). In stored plasma samples, the assay performance was set by design to have 100% sensitivity, which resulted in 82% specificity for ciHHV-6.

Conclusions: The possibility of ciHHV-6 is often overlooked in patients with detectable HHV-6 viral loads by quantitative PCR. Our ddPCR test provides rapid and accurate laboratory identification of ciHHV-6 from easily obtained cellular samples. In addition, the assay provides excellent sensitivity and specificity using stored plasma samples, facilitating retrospective analysis of the clinical significance of ciHHV-6.

Figure 1

A. Droplet plot of positive and negative droplets for HHV-6/RPP30 duplex ddPCR assay using template DNA from Hector-2 ciHHV-6 positive control cells. Upper left dots are positive for the HHV-6 FAM assay, lower right dots are positive for the RPP30 HEX assay, upper right dots are positive for both assays and lower left dots are negative for both assays. B. Concordance of HHV-6 copy number with cell number in ciHHV-6 positive control Hector-2 cells assayed by the duplexed digital PCR assay for HHV-6 and RPP30 run in duplicate.

Figure 2

Dilution series 10-fold) of Hector-2 ciHHV-6 cell line indicates that the ddPCR assay provides a precise ratio of HHV-6/cell with as few as 10⁴ cells.

Figure 3

HHV-6/cell ddPCR results for plasma samples from hematopoietic stem cell transplant study patients with ciHHV-6 (ciHHV-6 +), reactivated HHV-6 infection (HHV-6 +), or no HHV-6 reactivation (HHV-6−).

Figure 4

HHV-6/cell ddPCR results for plasma samples from residual clinical specimens that were leftover from routine qPCR testing for HHV-6 viral load and were positive by qPCR (HHV-6+). Negative control samples (HHV-6 −) are leftover samples from routine qPCR testing for Human Cytomegalovirus (CMV).