 Collectively, these results demonstrate HHV-6 in the saliva of 67% (26/39) healthy donors. Of these HHV-6 positive saliva samples, only 12% (3/26) were found to be coinfected with HHV-6A and HHV-6B (summarized in Table 3), lower than what was observed in the PBMC (Figure 2) or serum (Figure 3). We did not observe correlations between coinfection and gender or ethnicity (data not shown). This high frequency of detection [19,21,40,41] and range of viral copies/ml saliva [20] are both consistent with other published reports.
Of the HHV-6 viruses, HHV-6A has been suggested to be more neurotropic than HHV-6B [33,42] and is associated with MS [43,44]. Therefore, we asked if the level of coinfection (which in this study is defined as the presence of HHV-6A in addition to HHV-6B) is increased among MS patients compared to healthy donors ( Figure 6D). The rates of viral detection in saliva were similar between MS patients (63%) and healthy donors (67%),  with a mean total viral load of 1.82610 4 copies/ml for MS patients and 1.38610 4 copies/ml for healthy donors (summarized in Table 3). However, there was a significantly elevated prevalence of coinfection in MS patient saliva (30% (11/37)) compared to the healthy controls (12% (3/26)) (X 2 test, p = 0.04) ( Figure 6E), suggesting that the difference between MS patients and controls may be the increased presence of HHV-6A in addition to HHV-6B. The amounts of HHV-6A and HHV-6B within each coinfected sample are shown in Figure 6F. The 6A/6B ratio of copies/ml saliva ranged from 0.005-0.6 in healthy donors and from 0.04 to 4.0 in MS patients, demonstrating that in healthy donors, all coinfected samples contained more HHV-6B compared to HHV-6A, while in MS patients, a subset of the coinfected samples contained more HHV-6A than HHV-6B (2/11). In this particular MS cohort, there was no correlation between coinfection and expanded disability status scale (EDSS) score, brain lesion load or contrast enhancing lesions around the time of saliva collection (data not shown).

Discussion
The HHV-6 viruses are detected at low levels in the majority of healthy individuals. This is supported by the widespread-but controversial-use of nested PCR to establish the frequency of HHV-6 in the blood of healthy donors [19], as well as data demonstrating that viral detection in blood increases with increasing cellular input [19,20,24]. Such low levels of virus render the detection of viral DNA highly subject to both the sample integrity and assay sensitivity, and may underlie discrepancies in the literature regarding the frequency of HHV-6 detection among healthy individuals.
In this paper, we report a novel HHV-6 detection method using a third generation quantitative PCR technology, ddPCR. DdPCR  Table 3. Summary of HHV-6 detection by ddPCR HHV-6 positive samples.  is ideal for detecting low frequency events, such as HHV-6 viral DNA in healthy donor samples, because it partitions a sample into discrete droplets, thereby minimizing random sampling error. Moreover, increasing the number of replicates proportionally increases the quantitative dynamic range; this ability to extend the lower limits of detection is ideal for assessing low-level targets [28], and important for detecting two viral species that may be present in different quantities. We detected HHV-6 viral DNA in the PBMC of 57% (26/46) of healthy donors, consistent with a number of previous studies [20,45]. When considering prevalence data in a cross-sectional study such as this one, it is important to note that detectable viral DNA levels within an individual may fluctuate over time. This was demonstrated in a longitudinal study of healthy children, which reported that over several years, viral DNA was detected intermittently in the PBMC of 76% children, but consistently detected in the PBMC of only 12% of children [46].
In our study of healthy donor PBMC, we fortuitously identified two individuals with probable CI HHV-6, one with HHV-6A and the other with HHV-6B, though analysis by fluorescent in situ hybridization (FISH) is necessary to confirm chromosomal integration. The precise mechanisms and clinical implications of chromosomally integrated HHV-6A and HHV-6B are active areas of study [47,48]. From studies of healthy blood donors in the US and UK, the incidence of HHV-6 chromosomal integration in the general population is thought to be approximately 1% [37]. We believe that the percentage of potentially chromosomally integrated individuals as calculated from this study (2/48, or 4%) reflects low numbers of surveyed PBMC.
In the healthy blood donor with probable integrated HHV-6A (1.02610 6 copies HHV-6A per 10 6 cells) we also detected HHV-6B viral DNA (306 copies HHV-6B per 10 6 cells). These data are consistent with a recent report of HHV-6B detection in two individuals determined to be CI HHV-6A [35]. Notably, we detected these HHV-6B positive droplets both when the sample was duplexed (306 copies HHV-6B/10 6 cells) and triplexed (401 copies HHV-6B/10 6 cells). These data highlight the suitability of ddPCR for assessing coinfection, as low copy numbers of one viral species can be detected and reproducibly quantified in the presence of excess copies of the other viral species.
The detection of viral nucleic acid in blood may reflect active viral replication, chronic persistence or latency [1]. We detected HHV-6 DNA in 30% (13/43) serum samples from healthy adult donors. These results are higher than many previous studies [21,[49][50][51][52] that employed conventional PCR approaches and reported undetectable levels of HHV-6 DNA in healthy donor serum. However, a frequency of 30% agrees with several other studies, including one that used a highly sensitive PCR ELISA assay and reported HHV-6 DNA in the serum of 21% (5/24) healthy Canadian donors [36] and another that reported 20% detection among healthy Jordanian controls [53]. It is currently thought that HHV-6 DNA detected in the serum indicates an active infection, but recent work cautions that viral DNA in the serum may in fact reflect DNA released from circulating blood cells [54]. While cellular material in serum can indicate the presence of genomic DNA contaminants [55], we found no correlation between viral copies/ml serum and the cellular housekeeping gene (a subset shown in Figure 4B), suggesting that viral DNA we detected in serum may not be cell-associated. An active infection can also be hypothesized based on the ratio of virus in the serum to virus in circulating peripheral blood cells ( Figure 4A). For example, most individuals in our study had a ratio of approximately one, but one individual had a ratio of 11. This 11-fold increase of virus in the serum compared to PBMC supports further studies on this individual to determine whether there is active or infectious virus in this compartment.
A major finding of this report is the high frequency of HHV-6A and HHV-6B coinfection in healthy adult donors. There are few published studies on coinfection of the HHV-6 viruses, likely because many early reports did not distinguish between HHV-6A and HHV-6B, though the literature is shifting towards this distinction [56]. Even in studies that report coinfection [21,57], there may be an underestimation of the frequency. For example, in one study of 44 adult PBMC samples, coinfection was detected in 18% of the HHV-6 positive samples [57]. This is comparatively lower than our data, as we detected coinfection in 50% (13/26) of HHV-6 positive PBMC samples. We also report coinfection in 62% (8/13) of HHV-6 positive serum samples, and are not aware of other studies that observed this in healthy donor serum. This high percentage likely reflects the low denominator, and should be interpreted with caution.
In addition to PBMC and serum, we also analyzed the HHV-6A and HHV-6B viral loads in saliva. The salivary glands are a known reservoir for HHV-6 [38] and saliva is an easily collected clinical sample. Among healthy donors, we observed the lowest incidence of HHV-6A and HHV-6B coinfection in this compartment (12%, relative to 50% in PBMC and 62% in serum). However, this observation may be population-specific, as a recent study of HHV-6 in the saliva of healthy Brazilian donors reported twice the frequency of HHV-6A compared to HHV-6B [25].
As we observed higher viral loads in the saliva compared to the blood of healthy donors, we chose this sample type to compare HHV-6 levels between healthy donors and patients with MS, a neurologic disease associated with HHV-6 (reviewed in [58]). Despite comparable detection frequencies and total viral loads between healthy donor and MS saliva, we detected significantly increased HHV-6A and HHV-6B coinfection in the MS samples. These data extend observations from a previous study in which we detected coinfection in a subset of HHV-6 positive saliva samples from MS patients (6