Digital Droplet PCR Assay for HHV-6 shows promise for detecting low-grade infection and identifying ciHHV-6
Two papers published this month highlight the potential of the new 3rd generation PCR assays for samples with very low viral loads (MS patient sera) and for identifying ciHHV-6 in PBMCs. This new assay uses proprietary technology to fractionate the sample into 20,000 “water-in-oil” emulsion droplets and PCR amplification occurs in each individual droplet, resulting in an assay that is far more precise than traditional qPCR.
In a paper published this month in PLOS One (Liebovitch 2014), Steve Jacobson’s group at NINDS/NIH used techniques to increase the sensitivity of the ddPCR assay, allowing an exploration of low copy number viruses such as HHV-6 in MS serum. Although levels of HHV-6A and HHV-6B DNA typically fall below the level of detection in all but acutely ill patients (or those with ciHHV-6), Jacobson’s group found HHV-6 DNA in 30% of the serum and 57% of the PBMCs of healthy controls. This is a far higher prevalence than is achieved in other recent studies. In addition, HHV-6B levels were found to be consistently higher than HHV-6A levels. The study also found that MS patients had significantly more HHV-6A detectable in saliva than healthy controls (30% vs. 12%) as well as a higher viral load. HHV-6A is considered more neurotropic and is the virus species implicated as a potential trigger in some MS patients.
Since HHV-6 is both a low copy number virus and highly cell-associated, investigators looking for HHV-6 DNA in MS patients must use nested PCR or ultrasensitive assays when testing plasma or serum. Often, the HHV-6 DNA can often only be found at the time of a “flare-up.” MS investigators Roberto Alvarez-Lafuente in Spain, Mauro Malnati in Italy, and Steve Jacobson in the USA typically use larger sample sizes (1-2 ml of material) and further concentrate the source material using a high-speed centrifuge in an effort to increase sensitivity. In the present study, Jacobson’s team used 1.0 ml of serum and the QuiAmp ultrasensitive Virus Kit for his ddPCR assay. This kit uses a novel technology to concentrate viral nucleic acids in plasma and serum samples; the reagent forms complexes with nucleic acids allowing them to be highly concentrated by low-speed centrifugation.
Jacobson’s group compared serum and PBMC viral loads and found that in most cases, they do not correlate, suggesting that the DNA in serum may originate from compartments other than the peripheral blood. This finding is similar to what has been previously described by Nitcshe et al in 2001, in one of the few studies that investigated both HHV-6A and HHV-6B simultaneously in both PBMCs and plasma samples. HHV-6A was found in 92% of plasma samples but only 4% of PBMC samples, suggesting to the authors that the HHV-6A may have originated from compartments outside of the circulating peripheral blood.
Keith Jerome’s group at the University of Washington has also begun utilizing ddPCR technology in the field of HHV-6 diagnostics. In a study published this month, their team has reported the validation of an assay designed to confirm ciHHV-6 status using cellular samples (Sedlak 2014). The group compared HHV-6 DNA levels to the number of cells and found that ciHHV-6 patients produced a genome-to-cell ratio very close to 1:1. The University of Washington assay was not designed to be ultra-sensitive, however, and does not differentiate between the ciHHV-6A and ciHHV-6B viruses.
Unfortunately, another limitation of this assay is its inability to definitively determine ciHHV-6 status from a plasma sample, and thus the authors recommend that cellular samples be used to determine ciHHV-6 status whenever possible. They also noted that ciHHV-6 patients can have lower viral load levels in acellular material than previously believed (>3,500 copies/ml), and that the viral load in plasma or serum ciHHV-6 samples varies greatly depending on the amount of time between blood draw and centrifuge. This is likely because as more time elapses between the draw and centrifugation, more cells have the opportunity to lyse, and thus more integrated DNA is released into the plasma or serum.
For more information, read the full papers here: