Detection of Human Herpesvirus 6B (HHV-6B) Reactivation in Hematopoietic Cell Transplant Recipients with Inherited Chromosomally Integrated HHV-6A by Droplet Digital PCR

Abstract

The presence of inherited chromosomally integrated human herpesvirus 6 (ciHHV-6) in hematopoietic cell transplant (HCT) donors or recipients confounds molecular testing for HHV-6 reactivation, which occurs in 30 to 50% of transplants. Here we describe a multiplex droplet digital PCR clinical diagnostic assay that concurrently distinguishes between HHV-6 species (A or B) and identifies inherited ciHHV-6. By applying this assay to recipient post-HCT plasma and serum samples, we demonstrated reactivation of HHV-6B in 25% (4/16 recipients) of HCT recipients with donor- or recipient-derived inherited ciHHV-6A, underscoring the need for diagnostic testing for HHV-6 infection even in the presence of ciHHV-6.

FIG 1

A ddPCR multiplex assay for HHV-6A, HHV-6B, and the cell reference gene RPP30 identifies inherited ciHHV-6 (iciHHV-6) and distinguishes HHV-6A from HHV-6B in human whole-blood, serum, and plasma samples. (A) ddPCR droplet plot showing HHV-6A (blue box)- and HHV-6B (red box)-positive droplet populations at different x and y amplitudes. Gray droplets are negative for template DNA. Green droplets are positive for RPP30. Blue droplets are positive for HHV-6A or -B. Orange droplets are positive for both RPP30 and HHV-6A or -B. (B) Concordance between the non-species-discriminating iciHHV-6 ddPCR assay and the iciHHV-6A/B ddPCR assay. (C) Data for a negative-control serum spiked with a ciHHV-6A control cell line (squares) and a 10-fold dilution series of HHV-6B particles (open circles). Bars represent the standard deviations of the means for 3 ddPCRs. (D) Mean HHV-6A loads for three separate classes of iciHHV-6A HCT recipients: recipient integrated (n = 6; filled boxes), donor integrated (n = 7; filled circles), and both donor and recipient integrated (n = 3; open triangles).

FIG 2

Multiplex iciHHV-6A/B ddPCR sensitively and simultaneously detects HHV-6B and ciHHV-6A in serum samples from four individual HCT recipients. Sera of ciHHV-6A HCT recipients were tested for reactivation of HHV-6A (squares) and HHV-6B (open circles). Each point represents the mean for 3 ddPCRs. The dashed line at 10² copies/ml represents the limit of detection for HHV-6B. Each asterisk (*) denotes a sample positive for HHV-6B by qualitative real-time PCR. (A, C, and D) Three individual HCT recipients with donor-derived ciHHV-6A; (B) HCT recipient with related donor- and recipient-derived ciHHV-6A.